Publications (1999–2016)

For publications dating back to 1987, visit Google Scholar.

@article{pmid26524397,
  abstract = { Molecular machines have previously been designed that are propelled by DNAzymes, protein enzymes and strand displacement. These engineered machines typically move along precisely defined one- and two-dimensional tracks. Here, we report a DNA walker that uses hybridization to drive walking on DNA-coated microparticle surfaces. Through purely DNA:DNA hybridization reactions, the nanoscale movements of the walker can lead to the generation of a single-stranded product and the subsequent immobilization of fluorescent labels on the microparticle surface. This suggests that the system could be of use in analytical and diagnostic applications, similar to how strand exchange reactions in solution have been used for transducing and quantifying signals from isothermal molecular amplification assays. The walking behaviour is robust and the walker can take more than 30 continuous steps. The traversal of an unprogrammed, inhomogeneous surface is also due entirely to autonomous decisions made by the walker, behaviour analogous to amorphous chemical reaction network computations, which have been shown to lead to pattern formation. },
  month = { Feb },
  pages = { 157--163 },
  number = { 2 },
  volume = { 11 },
  year = { 2016 },
  journal = { Nat Nanotechnol },
  title = { {A} stochastic {D}{N}{A} walker that traverses a microparticle surface },
  author = { Jung and Allen and Ellington },
}

@article{pmid26750592,
  abstract = { During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a "competitive" aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform. },
  month = { Feb },
  pages = { 2250--2257 },
  number = { 4 },
  volume = { 88 },
  year = { 2016 },
  journal = { Anal. Chem. },
  title = { {E}ngineering {S}ignaling {A}ptamers {T}hat {R}ely on {K}inetic {R}ather {T}han {E}quilibrium {C}ompetition },
  author = { Du and Zhen and Li and Byrom and Jiang and Ellington },
}

@article{pmid26780407,
  abstract = { Engineered orthogonal translation systems have greatly enabled the expansion of the genetic code using noncanonical amino acids (NCAAs). However, the impact of NCAAs on organismal evolution remains unclear, in part because it is difficult to force the adoption of new genetic codes in organisms. By reengineering TEM-1 β-lactamase to be dependent on a NCAA, we maintained bacterial NCAA dependence for hundreds of generations without escape. },
  month = { Mar },
  pages = { 138--140 },
  number = { 3 },
  volume = { 12 },
  year = { 2016 },
  journal = { Nat. Chem. Biol. },
  title = { {A}ddicting diverse bacteria to a noncanonical amino acid },
  author = { Tack and Ellefson and Thyer and Wang and Gollihar and Forster and Ellington },
}

@article{pmid26844430,
  abstract = { High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample. },
  month = { Mar },
  pages = { 429--442 },
  number = { 3 },
  volume = { 11 },
  year = { 2016 },
  journal = { Nat Protoc },
  title = { {U}ltra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes },
  author = { McDaniel and DeKosky and Tanno and Ellington and Georgiou },
}

@article{pmid26899430,
  abstract = { The selection of RNA and DNA aptamers now has a long history. However, the ability to directly select for conformational changes upon ligand binding has remained elusive. These difficulties have stymied attempts at making small molecule responsive strand displacement circuitry as well as synthetic riboswitches. Herein we present a detailed strand displacement based selection protocol to directly select for RNA molecules with switching activity. The library was based on a previously selected thiamine pyrophosphate riboswitch. The fully in vitro methodology gave sequences that showed strong strand displacement activity in the presence of thiamine pyrophosphate. Further, the selected sequences possessed riboswitch activity similar to that of natural riboswitches. The presented methodology should aid in the design of more complex, environmentally responsive strand displacement circuitry and in the selection of riboswitches responsive to toxic ligands. },
  month = { Feb },
  pages = {   },
  year = { 2016 },
  journal = { Methods },
  title = { {A}n in vitro selection for small molecule induced switching {R}{N}{A} molecules },
  author = { Martini and Ellington and Mansy },
}

@article{pmid27032410,
  abstract = { There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3' terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays. Graphical abstract Efficient self-folding by phosphorothioate (PS) modification. },
  month = { Mar },
  pages = {   },
  year = { 2016 },
  journal = { Anal Bioanal Chem },
  title = { {A} primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension ({P}{S}-{T}{H}{S}{P}) },
  author = { Jung and Ellington },
}

@article{pmid27114511,
  abstract = { Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease. },
  month = { May },
  pages = { E2636--2645 },
  number = { 19 },
  volume = { 113 },
  year = { 2016 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {L}arge-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires },
  author = { DeKosky and Lungu and Park and Johnson and Charab and Chrysostomou and Kuroda and Ellington and Ippolito and Gray and Georgiou },
}

@article{pmid27189550,
  abstract = { Until recently, evolutionary questions surrounding the nature of the genetic code have been mostly limited to the realm of conjecture, modeling, and simulation due to the difficulty of altering this fundamental property of living organisms. Concerted genome and protein engineering efforts now make it possible to experimentally study the impact of alternative genetic codes on the evolution of biological systems. We explored how Escherichia coli strains that incorporate a 21(st) nonstandard amino acid (nsAA) at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. Resistance to rifampicin arises from chromosomal mutations in the β subunit of RNA polymerase (RpoB). We found that a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance. We interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways: by adding a new letter with unique chemical properties to the protein alphabet and by altering the mutational connectivity of amber-adjacent codons by converting a lethal nonsense mutation into a missense mutation. Finally, we consider the implications of these results for the evolution of alternative genetic codes. In our experiments, reliance on a mutation to a reassigned codon for a vital trait is not required for the long-term maintenance of an expanded genetic code and may even destabilize incorporation of a nsAA, a result that is consistent with the codon capture model of genetic code evolution. },
  month = { May },
  pages = {   },
  year = { 2016 },
  journal = { Mol. Biol. Evol. },
  title = { {E}xpanded genetic codes create new mutational routes to rifampicin resistance in {E}scherichia coli },
  author = { Hammerling and Gollihar and Mortensen and Alnahhas and Ellington and Barrick },
}

@article{pmid25265557,
  abstract = { The economic theory of comparative advantage postulates that beneficial trading relationships can be arrived at by two self-interested entities producing the same goods as long as they have opposing relative efficiencies in producing those goods. The theory predicts that upon entering trade, in order to maximize consumption both entities will specialize in producing the good they can produce at higher efficiency, that the weaker entity will specialize more completely than the stronger entity, and that both will be able to consume more goods as a result of trade than either would be able to alone. We extend this theory to the realm of unicellular organisms by developing mathematical models of genetic circuits that allow trading of a common good (specifically, signaling molecules) required for growth in bacteria in order to demonstrate comparative advantage interactions. In Conception 1, the experimenter controls production rates via exogenous inducers, allowing exploration of the parameter space of specialization. In Conception 2, the circuits self-regulate via feedback mechanisms. Our models indicate that these genetic circuits can demonstrate comparative advantage, and that cooperation in such a manner is particularly favored under stringent external conditions and when the cost of production is not overly high. Further work could involve implementing the models in living bacteria and searching for naturally occurring cooperative relationships between bacteria that conform to the principles of comparative advantage. },
  month = { Jan },
  pages = { 326--343 },
  volume = { 364 },
  year = { 2015 },
  journal = { J. Theor. Biol. },
  title = { {A} microbial model of economic trading and comparative advantage },
  author = { Enyeart and Simpson and Ellington },
}

@article{pmid25279711,
  abstract = { T7 RNA polymerase is the foundation of synthetic biological circuitry both in vivo and in vitro due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel. },
  month = { Oct },
  pages = { 1070--1076 },
  number = { 10 },
  volume = { 4 },
  year = { 2015 },
  journal = { ACS Synth Biol },
  title = { {D}irected {E}volution of a {P}anel of {O}rthogonal {T}7 {R}{N}{A} {P}olymerase {V}ariants for in {V}ivo or in {V}itro {S}ynthetic {C}ircuitry },
  author = { Meyer and Ellefson and Ellington },
}

@article{pmid25501908,
  abstract = { High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza. },
  month = { Jan },
  pages = { 86--91 },
  number = { 1 },
  volume = { 21 },
  year = { 2015 },
  journal = { Nat. Med. },
  title = { {I}n-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire },
  author = { DeKosky and Kojima and Rodin and Charab and Ippolito and Ellington and Georgiou },
}

@article{pmid25521771,
  abstract = { Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNA(Sec) needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNA(Sec) that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNA(Sec) allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds. },
  month = { Jan },
  pages = { 46--49 },
  number = { 1 },
  volume = { 137 },
  year = { 2015 },
  journal = { J. Am. Chem. Soc. },
  title = { {E}volving t{R}{N}{A}({S}ec) for efficient canonical incorporation of selenocysteine },
  author = { Thyer and Robotham and Brodbelt and Ellington },
}

@article{pmid25603321,
  abstract = { DNA-protein conjugates have found a wide range of applications. This study demonstrates the formation of defined, non-native protein-protein complexes via the site specific labeling of two proteins of interest with complementary strands of single-stranded DNA in vitro. This study demonstrates that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation. },
  month = { Mar },
  pages = { 427--434 },
  number = { 3 },
  volume = { 26 },
  year = { 2015 },
  journal = { Bioconjug. Chem. },
  title = { {C}ontrolled assembly of artificial protein-protein complexes via {D}{N}{A} duplex formation },
  author = { P?osko? and Wagner and Ellington and Jewett and O'Reilly and Booth },
}

@article{pmid25605388,
  abstract = { We describe design parameters for the synthesis and analytical application of a label-free RNA molecular beacon, termed Spinach.ST. The RNA aptamer Spinach fluoresces upon binding the small-molecule fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one). Spinach has been reengineered by extending its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation that fails to bind DHFBI. Hybridization of a trigger oligonucleotide to a designed toehold on Spinach.ST initiates toehold-mediated strand displacement and restores the DFHBI-binding, fluorescence-enhancing conformation of Spinach. The versatile Spinach.ST sensor can detect DNA or RNA trigger sequences and can readily distinguish single-nucleotide mismatches in the trigger toehold. Primer design techniques are described that augment amplicons produced by enzymatic amplification with Spinach.ST triggers. Interaction between these triggers and Spinach.ST molecular beacons leads to the real-time, sequence-specific quantitation of these amplicons. The use of Spinach.ST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications. The same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans. },
  pages = { 215--249 },
  volume = { 550 },
  year = { 2015 },
  journal = { Meth. Enzymol. },
  title = { {D}esign, synthesis, and application of {S}pinach molecular beacons triggered by strand displacement },
  author = { Bhadra and Ellington },
}

@article{pmid25708458,
  abstract = { Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests. },
  month = { Mar },
  pages = { 3314--3320 },
  number = { 6 },
  volume = { 87 },
  year = { 2015 },
  journal = { Anal. Chem. },
  title = { {R}obust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons },
  author = { Jiang and Bhadra and Li and Wu and Milligan and Ellington },
}

@article{pmid25780159,
  month = { Apr },
  pages = { 608 },
  number = { 4 },
  volume = { 21 },
  year = { 2015 },
  journal = { RNA },
  title = { {R}{N}{A} as a conception },
  author = { Ellington },
}

@article{pmid25826754,
  abstract = { Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi-specific receptors, Next-Gen DNA sequencing was used to generate data, and cell line differentiation was the test-bed application. The study of a principal component analysis loading plot revealed cross-reactivity between the aptamers. The technique generates high-dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid-based receptors exist. },
  month = { May },
  pages = { 6339--6342 },
  number = { 21 },
  volume = { 54 },
  year = { 2015 },
  journal = { Angew. Chem. Int. Ed. Engl. },
  title = { {N}ext-generation sequencing as input for chemometrics in differential sensing routines },
  author = { Goodwin and Gade and Byrom and Herrera and Spears and Anslyn and Ellington },
}

@article{pmid25856093,
  abstract = { The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens. },
  pages = { e0123126 },
  number = { 4 },
  volume = { 10 },
  year = { 2015 },
  journal = { PLoS ONE },
  title = { {R}eal-time sequence-validated loop-mediated isothermal amplification assays for detection of {M}iddle {E}ast respiratory syndrome coronavirus ({M}{E}{R}{S}-{C}o{V}) },
  author = { Bhadra and Jiang and Kumar and Johnson and Hensley and Ellington },
}

@article{pmid25967118,
  abstract = { While DNA circuits are becoming increasingly useful as signal transducers, their utility is inhibited by their slow catalytic rate. Here, we demonstrate how RecA, a recombination enzyme that catalyzes sequence specific strand exchange, can be used to increase circuit rates up to 9-fold. We also show how the introduction of RNA into DNA circuits further controls the specificity of RecA strand exchange, improving signal-to-noise. },
  month = { Jun },
  pages = { 9503--9506 },
  number = { 46 },
  volume = { 51 },
  year = { 2015 },
  journal = { Chem. Commun. (Camb.) },
  title = { {U}sing {R}ec{A} protein to enhance kinetic rates of {D}{N}{A} circuits },
  author = { Milligan and Ellington },
}

@article{pmid25978303,
  abstract = { An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry. },
  month = { Oct },
  pages = { 1144--1150 },
  number = { 10 },
  volume = { 4 },
  year = { 2015 },
  journal = { ACS Synth Biol },
  title = { {I}n {V}itro {S}election for {S}mall-{M}olecule-{T}riggered {S}trand {D}isplacement and {R}iboswitch {A}ctivity },
  author = { Martini and Meyer and Ellefson and Milligan and Forlin and Ellington and Mansy },
}

@article{pmid25984570,
  abstract = { By relying on specific DNA:DNA interactions as a "smart glue", we have assembled microparticles into a colloidal gel that can hold its shape. This gel can be extruded with a 3D printer to generate centimeter size objects. We show four aspects of this material: (1) The colloidal gel material holds its shape after extrusion. (2) The connectivity among the particles is controlled by the binding behavior between the surface DNA and this mediates some control over the microscale structure. (3) The use of DNA-coated microparticles dramatically reduces the cost of DNA-mediated assembly relative to conventional DNA nanotechnologies and makes this material accessible for macroscale applications. (4) This material can be assembled under biofriendly conditions and can host growing cells within its matrix. The DNA-based control over organization should provide a new means of engineering bioprinted tissues. },
  month = { Jan },
  pages = { 19--26 },
  number = { 1 },
  volume = { 1 },
  year = { 2015 },
  journal = { ACS Biomater Sci Eng },
  title = { 3{D} {P}rinting with {N}ucleic {A}cid {A}dhesives },
  author = { Allen and Khaing and Schmidt and Ellington },
}

@article{pmid26050646,
  abstract = { Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device. },
  pages = { 11039 },
  volume = { 5 },
  year = { 2015 },
  journal = { Sci Rep },
  title = { {A} {S}weet {S}pot for {M}olecular {D}iagnostics: {C}oupling {I}sothermal {A}mplification and {S}trand {E}xchange {C}ircuits to {G}lucometers },
  author = { Du and Hughes and Bhadra and Jiang and Ellington and Li },
}

@article{pmid26201734,
  month = { Aug },
  pages = { 617--619 },
  number = { 8 },
  volume = { 7 },
  year = { 2015 },
  journal = { Nat Chem },
  title = { {S}ynthetic biology: {S}ix pack and stack },
  author = { Jung and Ellington },
}

@article{pmid26209133,
  abstract = { On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed. },
  month = { Sep },
  pages = { 7480--7488 },
  number = { 15 },
  volume = { 43 },
  year = { 2015 },
  journal = { Nucleic Acids Res. },
  title = { {T}ranscription yield of fully 2'-modified {R}{N}{A} can be increased by the addition of thermostabilizing mutations to {T}7 {R}{N}{A} polymerase mutants },
  author = { Meyer and Garry and Hall and Byrom and McDonald and Yang and Yin and Ellington },
}

@article{pmid26224395,
  abstract = { We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min. },
  pages = { 3707--3715 },
  number = { 18 },
  volume = { 15 },
  year = { 2015 },
  journal = { Lab Chip },
  title = { {P}aper diagnostic device for quantitative electrochemical detection of ricin at picomolar levels },
  author = { Cunningham and Scida and Kogan and Wang and Ellington and Crooks },
}

@article{pmid26332362,
  abstract = { Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts. },
  month = { Oct },
  pages = { 2405--2414 },
  number = { 10 },
  volume = { 10 },
  year = { 2015 },
  journal = { ACS Chem. Biol. },
  title = { C}hemical {T}ools {T}o {D}ecipher {R}egulation of {P}hosphatases by {P}roline {I}somerization on {E}ukaryotic {R}{N}{A} {P}olymerase {I}{I },
  author = { Mayfield and Fan and Wei and Zhang and Li and Ellington and Etzkorn and Zhang },
}

@article{pmid26355042,
  abstract = { The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138(+) antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported. },
  pages = { 13926 },
  volume = { 5 },
  year = { 2015 },
  journal = { Sci Rep },
  title = { {F}acile {D}iscovery of a {D}iverse {P}anel of {A}nti-{E}bola {V}irus {A}ntibodies by {I}mmune {R}epertoire {M}ining },
  author = { Wang and Kluwe and Lungu and DeKosky and Kerr and Johnson and Jung and Rezigh and Carroll and Reyes and Bentz and Villanueva and Altman and Davey and Ellington and Georgiou },
}

@article{pmid26355089,
  abstract = { Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat that they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. For example, a human single nucleotide polymorphism (SNP) in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable cocrystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step toward computationally predicting the "host jumping" potential of a virus into a new host species.\\ We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing the potential spillover risks of viruses circulating in animal populations. },
  month = { Nov },
  pages = { 11643--11653 },
  number = { 22 },
  volume = { 89 },
  year = { 2015 },
  journal = { J. Virol. },
  title = { {C}omputational and {F}unctional {A}nalysis of the {V}irus-{R}eceptor {I}nterface {R}eveals {H}ost {R}ange {T}rade-{O}ffs in {N}ew {W}orld {A}renaviruses },
  author = { Kerr and Jackson and Lungu and Meyer and Demogines and Ellington and Georgiou and Wilke and Sawyer },
}

@article{pmid26465114,
  abstract = { Evolutionary innovations that enable organisms to colonize new ecological niches are rare compared to gradual evolutionary changes in existing traits. We discovered that key mutations in the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia coli gained the ability to grow aerobically on citrate (Cit(+) phenotype) during the Lenski long-term evolution experiment. The first gltA mutation, which increases CS activity by disrupting NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to preserving the rudimentary Cit(+) trait from extinction when it first evolved. However, after Cit(+) was refined by further mutations, this potentiating gltA mutation became deleterious to fitness. A second wave of beneficial gltA mutations then evolved that reduced CS activity to below the ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new nutrient niche contingent on both co-opting and overcoming a history of prior adaptation. },
  pages = {   },
  volume = { 4 },
  year = { 2015 },
  journal = { Elife },
  title = { {F}ine-tuning citrate synthase flux potentiates and refines metabolic innovation in the {L}enski evolution experiment },
  author = { Quandt and Gollihar and Blount and Ellington and Georgiou and Barrick },
}

@article{pmid26471233,
  abstract = { The advancement of synthetic biology over the past decade has contributed substantially to the growing bioeconomy. A recent report by the National Academies highlighted several areas of advancement that will be needed for further expansion of industrial biotechnology, including new focuses on design, feedstocks, processing, organism development, and tools for testing and measurement; more particularly, a focus on expanded chassis and end-to-end design in an effort to move beyond the use of E. coli and S. cerivisiea to organisms better suited to fermentation and production; second, continued efforts in systems biology and high-throughput screening with a focus on more rapid techniques that will provide the needed information for moving to larger scale; and finally, work to accelerate the building of a holacratic community with collaboration and engagement between the relevant government agencies, industry, academia, and the public. },
  month = { Oct },
  pages = { 1053--1055 },
  number = { 10 },
  volume = { 4 },
  year = { 2015 },
  journal = { ACS Synth Biol },
  title = { {I}ndustrialization of {B}iology },
  author = { Friedman and Ellington },
}

@article{pmid26480829,
  month = { Dec },
  pages = { 144--145 },
  number = { 5-6 },
  volume = { 81 },
  year = { 2015 },
  journal = { J. Mol. Evol. },
  title = { {L}andscape-{B}ased {B}iology },
  author = { Ellington },
}

@article{pmid26753172,
  abstract = { In this paper, we report a one-step tumor cell detection approach based on the dynamic morphological behavior tracking of cancer cells on a ligand modified surface. Every cell on the surface was tracked in real time for several minutes immediately after seeding until these were finally attached. Cancer cells were found to be very active in the aptamer microenvironment, changing their shapes rapidly from spherical to semi-elliptical, with much flatter spread and extending pseudopods at regular intervals. When incubated on a functionalized surface, the balancing forces between cell surface molecules and the surface-bound aptamers, together with the flexibility of the membranes, caused cells to show these distinct dynamic activities and variations in their morphologies. On the other hand, healthy cells remained distinguishingly inactive on the surface over the same period. The quantitative image analysis of cell morphologies provided feature vectors that were statistically distinct between normal and cancer cells. },
  month = { Dec },
  pages = { 194--200 },
  number = { 4 },
  volume = { 3 },
  year = { 2015 },
  journal = { Technology (Singap World Sci) },
  title = { {O}ne-step tumor detection from dynamic morphology tracking on aptamer-grafted surfaces },
  author = { Mahmood and Hasan and Khan and Allen and Kim and Ellington and Iqbal },
}

@article{pmid23680795,
  abstract = { Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes. },
  month = { Feb },
  pages = { 215--222 },
  number = { 2 },
  volume = { 185 },
  year = { 2014 },
  journal = { J. Struct. Biol. },
  title = { {S}tructure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex },
  author = { Xu and Tack and Hughes and Ellington and Gray },
}

@article{pmid24135653,
  abstract = { We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5'-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles. },
  month = { Jan },
  pages = { 38--40 },
  volume = { 445 },
  year = { 2014 },
  journal = { Anal. Biochem. },
  title = { {G}-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons },
  author = { Bhadra and Codrea and Ellington },
}

@article{pmid24185096,
  abstract = { Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression. },
  month = { Jan },
  pages = { 97--101 },
  number = { 1 },
  volume = { 32 },
  year = { 2014 },
  journal = { Nat. Biotechnol. },
  title = { {D}irected evolution of genetic parts and circuits by compartmentalized partnered replication },
  author = { Ellefson and Meyer and Hughes and Cannon and Brodbelt and Ellington },
}

@article{pmid24375025,
  abstract = { We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications. },
  month = { Jun },
  pages = { 1071--1081 },
  number = { 6 },
  volume = { 111 },
  year = { 2014 },
  journal = { Biotechnol. Bioeng. },
  title = { {D}irected evolution of the substrate specificity of biotin ligase },
  author = { Lu and Levy and Kincaid and Ellington },
}

@article{pmid24379390,
  abstract = { Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit(+)) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit(+) variants achieve a much higher population density in this environment. All Cit(+) individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit(+) phenotype on its own. To determine which of the other >70 mutations in early Cit(+) clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit(+) from an evolved strain. We discovered a mutation that increased expression of the dctA C4-dicarboxylate transporter greatly enhanced the Cit(+) phenotype after it evolved. Surprisingly, strains containing just the citT and dctA mutations fully use citrate, indicating that earlier mutations thought to have potentiated the initial evolution of Cit(+) are not required for expression of the refined version of this trait. Instead, this metabolic innovation may be contingent on a genetic background, and possibly ecological context, that enabled citT mutants to persist among competitors long enough to obtain dctA or equivalent mutations that conferred an overwhelming advantage. More generally, refinement of an emergent trait from a rudimentary form may be crucial to its evolutionary success. },
  month = { Feb },
  pages = { 2217--2222 },
  number = { 6 },
  volume = { 111 },
  year = { 2014 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {R}ecursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in {E}scherichia coli },
  author = { Quandt and Deatherage and Ellington and Georgiou and Barrick },
}

@article{pmid24402831,
  abstract = { Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can potentially react non-specifically even in the absence of a single-stranded catalyst, and this non-specific background degrades the signal-to-noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100-fold increased signal-to-background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non-enzymatic nucleic acid circuits. },
  month = { Feb },
  pages = { 1845--1848 },
  number = { 7 },
  volume = { 53 },
  year = { 2014 },
  journal = { Angew. Chem. Int. Ed. Engl. },
  title = { {M}ismatches improve the performance of strand-displacement nucleic {A}cid circuits },
  author = { Jiang and Bhadra and Li and Ellington },
}

@article{pmid24410776,
  abstract = { Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology. },
  pages = { 2 },
  number = { 1 },
  volume = { 5 },
  year = { 2014 },
  journal = { Mob DNA },
  title = { {B}iotechnological applications of mobile group {I}{I} introns and their reverse transcriptases: gene targeting, {R}{N}{A}-seq, and non-coding {R}{N}{A} analysis },
  author = { Enyeart and Mohr and Ellington and Lambowitz },
}

@article{pmid24413256,
  abstract = { Some metabolic pathway enzymes are known to organize into multi-enzyme complexes for reasons of catalytic efficiency, metabolite channeling, and other advantages of compartmentalization. It has long been an appealing prospect that de novo purine biosynthesis enzymes form such a complex, termed the "purinosome." Early work characterizing these enzymes garnered scarce but encouraging evidence for its existence. Recent investigations led to the discovery in human cell lines of purinosome bodies-cytoplasmic puncta containing transfected purine biosynthesis enzymes, which were argued to correspond to purinosomes. New discoveries challenge both the functional and physiological relevance of these bodies in favor of protein aggregation. },
  month = { Mar },
  pages = { 369--374 },
  number = { 3 },
  volume = { 10 },
  year = { 2014 },
  journal = { Mol Biosyst },
  title = { {R}evisiting and revising the purinosome },
  author = { Zhao and Tsechansky and Ellington and Marcotte },
}

@article{pmid24436411,
  month = { Jan },
  pages = { 259--260 },
  number = { 6168 },
  volume = { 343 },
  year = { 2014 },
  journal = { Science },
  title = { {B}iochemistry. {M}any paths to the origin of life },
  author = { Gollihar and Levy and Ellington },
}

@article{pmid24469811,
  abstract = { Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10(-8)-10(-10) M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3(-)CD14(-)CD19(+)CD27(++)CD38(++)CD20(-)TT(+)) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation. },
  month = { Feb },
  pages = { 2259--2264 },
  number = { 6 },
  volume = { 111 },
  year = { 2014 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {I}dentification and characterization of the constituent human serum antibodies elicited by vaccination },
  author = { Lavinder and Wine and Giesecke and Ippolito and Horton and Lungu and Hoi and DeKosky and Murrin and Wirth and Ellington and Dorner and Marcotte and Boutz and Georgiou },
}

@article{pmid24487692,
  abstract = { Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a noncanonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquire neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability. },
  month = { Mar },
  pages = { 178--180 },
  number = { 3 },
  volume = { 10 },
  year = { 2014 },
  journal = { Nat. Chem. Biol. },
  title = { {B}acteriophages use an expanded genetic code on evolutionary paths to higher fitness },
  author = { Hammerling and Ellefson and Boutz and Marcotte and Ellington and Barrick },
}

@article{pmid24488121,
  abstract = { Many normally cytosolic yeast proteins form insoluble intracellular bodies in response to nutrient depletion, suggesting the potential for widespread protein aggregation in stressed cells. Nearly 200 such bodies have been found in yeast by screening libraries of fluorescently tagged proteins. In order to more broadly characterize the formation of these bodies in response to stress, we employed a proteome-wide shotgun mass spectrometry assay in order to measure shifts in the intracellular solubilities of endogenous proteins following heat stress. As quantified by mass spectrometry, heat stress tended to shift the same proteins into insoluble form as did nutrient depletion; many of these proteins were also known to form foci in response to arsenic stress. Affinity purification of several foci-forming proteins showed enrichment for co-purifying chaperones, including Hsp90 chaperones. Tests of induction conditions and co-localization of metabolic enzymes participating in the same metabolic pathways suggested those foci did not correspond to multi-enzyme organizing centers. Thus, in yeast, the formation of stress bodies appears common across diverse, normally diffuse cytoplasmic proteins and is induced by multiple types of cell stress, including thermal, chemical, and nutrient stress. },
  month = { Apr },
  pages = { 851--861 },
  number = { 4 },
  volume = { 10 },
  year = { 2014 },
  journal = { Mol Biosyst },
  title = { {A} proteomic survey of widespread protein aggregation in yeast },
  author = { O'Connell and Tsechansky and Royall and Boutz and Ellington and Marcotte },
}

@article{pmid24493736,
  abstract = { Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer 'Spinach' that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits. },
  month = { Apr },
  pages = { e58 },
  number = { 7 },
  volume = { 42 },
  year = { 2014 },
  journal = { Nucleic Acids Res. },
  title = { {D}esign and application of cotranscriptional non-enzymatic {R}{N}{A} circuits and signal transducers },
  author = { Bhadra and Ellington },
}

@article{pmid24510437,
  abstract = { This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer-less PCR. The resulting chimeric genes can then be screened or selected for a desired function. },
  pages = { Unit 15.12. },
  volume = { 105 },
  year = { 2014 },
  journal = { Curr Protoc Mol Biol },
  title = { {L}ibrary generation by gene shuffling },
  author = { Meyer and Ellefson and Ellington },
}

@article{pmid24624315,
  abstract = { In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein-protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host-virus protein-protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein-protein interactions. },
  pages = { e266 },
  volume = { 2 },
  year = { 2014 },
  journal = { PeerJ },
  title = { {A}nalyzing machupo virus-receptor binding by molecular dynamics simulations },
  author = { Meyer and Sawyer and Ellington and Wilke },
}

@article{pmid24625092,
  abstract = { Cell-free systems are showing increasing promise for biosynthesis of both proteins and small molecules. However, in vitro transcription and translation reactions have so far primarily been used for the production of single proteins. In order to demonstrate the possibilities for coupled reactions, we designed synthetic operons that included different combinations of wild-type or evolved biotin ligases and streptavidins and demonstrated a mechanism for self-selection of operons following expression in vitro. Peptide substrates for biotin ligase were conjugated to the DNA operons and could be modified by a biotin ligase specific for either biotin or desthiobiotin and subsequently captured via a streptavidin specific for either biotin or desthiobiotin. },
  month = { Jun },
  pages = { 410--415 },
  number = { 6 },
  volume = { 3 },
  year = { 2014 },
  journal = { ACS Synth Biol },
  title = { {D}esign and selection of a synthetic operon },
  author = { Lu and Ellington },
}

@article{pmid24631721,
  abstract = { Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies. },
  month = { May },
  pages = { 12--22 },
  volume = { 178 },
  year = { 2014 },
  journal = { J. Biotechnol. },
  title = { {A}n amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins },
  author = { Singh-Blom and Hughes and Ellington },
}

@article{pmid24754567,
  abstract = { The purpose of this study was to compare the binding affinity and selective targeting of aptamer- and antibody-coated hollow gold nanospheres (HAuNS) targeted to epidermal growth factor receptors (EGFR). EGFR-targeting aptamers were conjugated to HAuNS (apt-HAuNS) by attaching a thiol-terminated single-stranded DNA to the HAuNS and then adding the complementary RNA targeted to EGFR. Apt-HAuNS was characterized in terms of size, surface charge, absorption, and number of aptamers per particle. The in vivo pharmacokinetics, in vivo biodistribution, and micro-SPECT/CT imaging of (111)In-labeled apt-HAuNS and anti-EGFR antibody (C225)-conjugated HAuNS were evaluated in nude mice bearing highly malignant human OSC-19 oral tumors. (111)In-labeled PEG-HAuNS was used as a control (n = 5/group). Apt-HAuNS did not have an altered absorbance profile or size (λmax = 800 nm; diameter = 55 nm) compared to C225-HAuNS or PEG-HAuNS. The surface charge became more negative upon conjugation of the aptamer (-51.4 vs -19.0 for PEG-HAuNS and -25.0 for C225-HAuNS). The number of aptamers/particle was ∼250. In vitro cell binding and in vivo biodistribution showed selective binding of the apt-HAuNS to EGFR. μSPECT/CT imaging confirmed that there was more tumor uptake of apt-HAuNS than C225-HAuNS. Aptamer is a promising ligand for image-guided delivery of nanoparticles for treatment of tumor cells overexpressing EGFR. },
  month = { May },
  pages = { 4530--4538 },
  number = { 5 },
  volume = { 8 },
  year = { 2014 },
  journal = { ACS Nano },
  title = { {S}elective uptake and imaging of aptamer- and antibody-conjugated hollow nanospheres targeted to epidermal growth factor receptors overexpressed in head and neck cancer },
  author = { Melancon and Zhou and Zhang and Xiong and Allen and Wen and Huang and Wallace and Myers and Stafford and Liang and Ellington and Li },
}

@article{pmid24828239,
  abstract = { CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options. },
  month = { Jun },
  pages = { 1825--1835 },
  number = { 6 },
  volume = { 47 },
  year = { 2014 },
  journal = { Acc. Chem. Res. },
  title = { {D}iagnostic applications of nucleic acid circuits },
  author = { Jung and Ellington },
}

@article{pmid24942625,
  abstract = { We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach. },
  month = { Aug },
  pages = { 1183--1194 },
  number = { 8 },
  volume = { 20 },
  year = { 2014 },
  journal = { RNA },
  title = { {A} {S}pinach molecular beacon triggered by strand displacement },
  author = { Bhadra and Ellington },
}

@article{pmid24990378,
  abstract = { The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a 'yes/no' answer. },
  month = { Sep },
  pages = { e120 },
  number = { 15 },
  volume = { 42 },
  year = { 2014 },
  journal = { Nucleic Acids Res. },
  title = { {E}xquisite allele discrimination by toehold hairpin primers },
  author = { Byrom and Bhadra and Jiang and Ellington },
}

@article{pmid25010201,
  abstract = { To make the electrochemical DNA sensors (E-sensor) more robust and reproducible, we have now for the first time adapted the techniques of ratiometric analyses to the field of E-sensors. We did this via the simple expedient way of simultaneously using two redox probes: Methylene blue as the reporter of the conformational change, and ferrocene as an internal control. During the conformational transduction, only the distance between the signal probe and the electrode surface undergoes an appreciable change, while the distance between the control probe and the electrode remains relatively constant. This special design has allowed very reliable target recognition, as illustrated in this report using a human T-lymphotropic virus type I gene fragment. The standard deviation between measurements obtained using different electrodes was an order of magnitude less than that obtained using a classic E-sensor, which we prepared as a control. A limit of detection of 25.1 pM was obtained with our new system, with a single mismatch discrimination factor of 2.33 likewise being observed. Additionally, this concept had general applicability, and preliminary data of a "Signal-On" ratiometric E-sensor are also provided. Taken in concert, these results serve to validate the utility of what we believe will emerge as an easily generalized approach to oligonucleotide recognition and sensing. },
  month = { Aug },
  pages = { 8010--8016 },
  number = { 15 },
  volume = { 86 },
  year = { 2014 },
  journal = { Anal. Chem. },
  title = { {R}eagentless, ratiometric electrochemical {D}{N}{A} sensors with improved robustness and reproducibility },
  author = { Du and Lim and Li and Jiang and Sessler and Ellington },
}

@article{pmid25080493,
  abstract = { Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa 'σ fragment', and fragments with different specificity can direct the remaining 601-aa 'core fragment' to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal 'α fragment' and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids. },
  pages = { 742 },
  volume = { 10 },
  year = { 2014 },
  journal = { Mol. Syst. Biol. },
  title = { {A} 'resource allocator' for transcription based on a highly fragmented {T}7 {R}{N}{A} polymerase },
  author = { Segall-Shapiro and Meyer and Ellington and Sontag and Voigt },
}

@article{pmid25506295,
  abstract = { We have developed a theoretical framework for developing patterns in multiple dimensions using controllable diffusion and designed reactions implemented in DNA. This includes so-called strand displacement reactions in which one single-stranded DNA hybridizes to a hemi-duplex DNA and displaces another single-stranded DNA, reversibly or irreversibly. These reactions can be designed to proceed with designed rate and molecular specificity. By also controlling diffusion by partial complementarity to a stationary, cross-linked DNA, we can generate predictable patterns. We demonstrate this with several simulations showing deterministic, predictable shapes in space. },
  month = { Dec },
  pages = { 583--595 },
  number = { 4 },
  volume = { 13 },
  year = { 2014 },
  journal = { Nat Comput },
  title = { {M}odeling {S}calable {P}attern {G}eneration in {D}{N}{A} {R}eaction {N}etworks },
  author = { Allen and Chen and Simpson and Ellington },
}

@article{pmid25606981,
  abstract = { Incorporation of modified nucleotides into in vitro RNA or DNA selections offers many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions. },
  pages = { 1--33 },
  volume = { 56 },
  year = { 2014 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {I}n vitro selection using modified or unnatural nucleotides },
  author = { Stovall and Bedenbaugh and Singh and Meyer and Hatala and Ellington and Hall },
}

@article{pmid22491026,
  abstract = { In vitro compartmentalization (IVC) is a method to generate numerous, small, aqueous compartments (up to 10(10) compartments per ml) by mixing water, surfactants, and oil. The water phase is surrounded by surfactants and an oil phase, and to a first approximation each water-in-oil compartment is like an artificial cell. By introducing single genes into compartments that are competent for transcription and translation, these cell-like compartments can synthesize RNA protein variants in libraries. Screening or selecting for function has in turn led to schemes for the directed evolution of biomolecules. However, IVC selections can cover larger library sizes, and provide greater control over selection conditions and stringencies. The key issue in designing and executing IVC selections is how to couple genotype and phenotype, and in this review we have organized and presented a variety of mechanisms by which proteins and RNA can attach to or amplify their own templates following emulsification and selection. },
  month = { Mar },
  pages = { 75--80 },
  number = { 1 },
  volume = { 60 },
  year = { 2013 },
  journal = { Methods },
  title = { {I}n vitro selection of proteins via emulsion compartments },
  author = { Lu and Ellington },
}

@article{pmid23104156,
  abstract = { Tumor cells depict two deviant tendencies; over-proliferation and vigorous migration. A tapered channel device is designed and fabricated for in vitro studies. We report inhibited proliferation and migration of human glioblastoma (hGBM) cells when exposed to an aptamer that is known to bind epidermal growth factor receptors (EGFR). The device is integrated with controlled ambient and microscope for providing real-time and quantitative characterization of the tumor cell behavior. The results show that hGBM cells loose proliferation and motility when exposed to the anti-EGFR aptamer. The aptamer directly inhibits and blocks EGF-induced EGFR phosphorylation. This also reduces the ability of cells to remodel their internal structure for invasion through narrow constrictions. This provides a framework for possible studies on efficacy of other inhibiting molecules. },
  month = { Aug },
  pages = { 635--643 },
  number = { 4 },
  volume = { 15 },
  year = { 2013 },
  journal = { Biomed Microdevices },
  title = { {P}roliferation and migration of tumor cells in tapered channels },
  author = { Wan and Tamuly and Allen and Kim and Bachoo and Ellington and Iqbal },
}

@article{pmid23334449,
  abstract = { Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination. },
  month = { Feb },
  pages = { 166--169 },
  number = { 2 },
  volume = { 31 },
  year = { 2013 },
  journal = { Nat. Biotechnol. },
  title = { {H}igh-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire },
  author = { DeKosky and Ippolito and Deschner and Lavinder and Wine and Rawlings and Varadarajan and Giesecke and Dorner and Andrews and Wilson and Hunicke-Smith and Willson and Ellington and Georgiou },
}

@article{pmid23382245,
  abstract = { We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease. },
  month = { Feb },
  pages = { 2993--2998 },
  number = { 8 },
  volume = { 110 },
  year = { 2013 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {M}olecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response },
  author = { Wine and Boutz and Lavinder and Miklos and Hughes and Hoi and Jung and Horton and Murrin and Ellington and Marcotte and Georgiou },
}

@article{pmid23405267,
  abstract = { It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation. },
  pages = { e56203 },
  number = { 2 },
  volume = { 8 },
  year = { 2013 },
  journal = { PLoS ONE },
  title = { {T}ransiently transfected purine biosynthetic enzymes form stress bodies },
  author = { Zhao and Tsechansky and Swaminathan and Cook and Ellington and Marcotte },
}

@article{pmid23423891,
  abstract = { The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino acid substitution on protein structure and function. Herein we describe protocols to introduce unnatural amino acids into proteins using the Escherichia coli translation system either in vivo or in vitro. Special attention is paid to obtaining high levels of incorporation while maintaining high yields of protein expression. },
  pages = { 93--114 },
  volume = { 978 },
  year = { 2013 },
  journal = { Methods Mol. Biol. },
  title = { {R}esidue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo },
  author = { Singh-Blom and Hughes and Ellington },
}

@article{pmid23509255,
  abstract = { Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular. },
  month = { Apr },
  pages = { 5386--5391 },
  number = { 14 },
  volume = { 110 },
  year = { 2013 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {S}tacking nonenzymatic circuits for high signal gain },
  author = { Chen and Briggs and McLain and Ellington },
}

@article{pmid23647466,
  abstract = { Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise. },
  month = { May },
  pages = { 7430--7433 },
  number = { 20 },
  volume = { 135 },
  year = { 2013 },
  journal = { J. Am. Chem. Soc. },
  title = { {R}eal-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly },
  author = { Jiang and Li and Milligan and Bhadra and Ellington },
}

@article{pmid23657394,
  month = { May },
  pages = { 413--415 },
  number = { 5 },
  volume = { 31 },
  year = { 2013 },
  journal = { Nat. Biotechnol. },
  title = { {E}ndowing cells with logic and memory },
  author = { Maranhao and Ellington },
}

@article{pmid23741319,
  abstract = { Reengineering protein surfaces to exhibit high net charge, referred to as "supercharging", can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from -11 to -61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and -30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding. },
  pages = { e64363 },
  number = { 5 },
  volume = { 8 },
  year = { 2013 },
  journal = { PLoS ONE },
  title = { {A}lternative computational protocols for supercharging protein surfaces for reversible unfolding and retention of stability },
  author = { Der and Kluwe and Miklos and Jacak and Lyskov and Gray and Georgiou and Ellington and Kuhlman },
}

@article{pmid23754529,
  abstract = { The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases. },
  month = { Jul },
  pages = { 471--478 },
  number = { 7 },
  volume = { 26 },
  year = { 2013 },
  journal = { Protein Eng. Des. Sel. },
  title = { {E}ngineered {D}{N}{A} ligases with improved activities in vitro },
  author = { Wilson and Morton and Deiderick and Gerth and Paul and Gerber and Patel and Ellington and Hunicke-Smith and Patrick },
}

@article{pmid23844594,
  abstract = { The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats, through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intramolecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 does not dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 toward the CTD peptide by 4-fold. To our surprise, even though the intramolecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription. },
  month = { Sep },
  pages = { 2042--2052 },
  number = { 9 },
  volume = { 8 },
  year = { 2013 },
  journal = { ACS Chem. Biol. },
  title = { novel modifications on {C}-terminal domain of {R}{N}{A} polymerase {I}{I} can fine-tune the phosphatase activity of {S}su72 },
  author = { Luo and Yogesha and Cannon and Yan and Ellington and Brodbelt and Zhang },
}

@article{pmid24002656,
  abstract = { Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%. },
  pages = { 685 },
  volume = { 9 },
  year = { 2013 },
  journal = { Mol. Syst. Biol. },
  title = { {G}eneralized bacterial genome editing using mobile group {I}{I} introns and {C}re-lox },
  author = { Enyeart and Chirieleison and Dao and Perutka and Quandt and Yao and Whitt and Keatinge-Clay and Lambowitz and Ellington },
}

@article{pmid24070108,
  abstract = { We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems. },
  month = { Oct },
  pages = { 9713--9720 },
  number = { 20 },
  volume = { 85 },
  year = { 2013 },
  journal = { Anal. Chem. },
  title = { {D}{N}{A} detection using origami paper analytical devices },
  author = { Scida and Li and Ellington and Crooks },
}

@article{pmid24214216,
  abstract = { Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule. },
  pages = { 433--442 },
  number = { 6 },
  volume = { 41 },
  year = { 2013 },
  journal = { Biochem Mol Biol Educ },
  title = { {C}ontinuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule },
  author = { Ledbetter and Hwang and Stovall and Ellington },
}

@article{pmid24256862,
  abstract = { Readily programmable chemical networks are important tools as the scope of chemistry expands from individual molecules to larger molecular systems. Although many complex systems are constructed using conventional organic and inorganic chemistry, the programmability of biological molecules such as nucleic acids allows for precise, high-throughput and automated design, as well as simple, rapid and robust implementation. Here we show that systematic and quantitative control over the diffusivity and reactivity of DNA molecules yields highly programmable chemical reaction networks (CRNs) that execute at the macroscale. In particular, we designed and implemented non-enzymatic DNA circuits capable of performing pattern-transformation algorithms such as edge detection. We also showed that it is possible to fine-tune and multiplex such circuits. We believe these strategies will provide programmable platforms on which to prototype CRNs, discover bottom-up construction principles and generate patterns in materials. },
  month = { Dec },
  pages = { 1000--1005 },
  number = { 12 },
  volume = { 5 },
  year = { 2013 },
  journal = { Nat Chem },
  title = { {P}attern transformation with {D}{N}{A} circuits },
  author = { Chirieleison and Allen and Simpson and Ellington and Chen },
}

@article{pmid21766299,
  abstract = { The detection of a small number of circulating tumor cells (CTCs) is important, especially in the early stages of cancer. Small numbers of CTCs are hard to detect, because very few approaches are sensitive enough to differentiate these from the pool of other cells. Improving the affinity of a selective, surface-functionalized molecule is important given the scarcity of CTCs in vivo. There are several proteins and aptamers that provide such high affinity; however, using surface nanotexturing increases this affinity even further.\\ The authors report an approach to improve the affinity of tumor cell capture by using novel aptamers against cell membrane overexpressed epidermal growth factor receptors (EGFRs) on a nanotextured polydimethylsiloxane (PDMS) substrate. Surface-immobilized aptamers were used to specifically capture tumor cells from physiologic samples.\\ The nanotexturing of PDMS increased surface roughness at the nanoscale. This increased the effective surface area and resulted in a significantly higher degree of surface functionalization. The phenomenon resulted in increased density of immobilized EGFR-specific RNA aptamer molecules and provided significantly higher efficiency to capture cancer cells from a mixture. The data indicated that CTCs could be captured and enriched, leading to higher yield yet higher background.\\ A comparison between glass slides, plain PDMS, and nanotextured PDMS functionalized with aptamers demonstrated that a 2-fold approach of using aptamers on nanotextured PDMS can be important for cancer cytology devices, and especially for the idea of a "lab-on-chip," toward higher yield in capture efficiency. },
  month = { Feb },
  pages = { 1145--1154 },
  number = { 4 },
  volume = { 118 },
  year = { 2012 },
  journal = { Cancer },
  title = { {N}anotextured substrates with immobilized aptamers for cancer cell isolation and cytology },
  author = { Wan and Mahmood and Li and Allen and Kim and Bachoo and Ellington and Iqbal },
}

@article{pmid22223052,
  abstract = { This article describes the development of a simple and robust fluorescence polarization (FP)-based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z' score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A. },
  month = { Apr },
  pages = { 448--459 },
  number = { 4 },
  volume = { 17 },
  year = { 2012 },
  journal = { J Biomol Screen },
  title = { {I}dentification of influenza virus inhibitors targeting {N}{S}1{A} utilizing fluorescence polarization-based high-throughput assay },
  author = { Cho and Xia and Ma and Robertus and Krug and Anslyn and Montelione and Ellington },
}

@article{pmid22233578,
  abstract = { We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells. },
  month = { Mar },
  pages = { 616--624 },
  number = { 3 },
  volume = { 20 },
  year = { 2012 },
  journal = { Mol. Ther. },
  title = { {A} general {R}{N}{A} motif for cellular transfection },
  author = { Magalhaes and Byrom and Yan and Kelly and Li and Furtado and Palliser and Ellington and Levy },
}

@article{pmid22520751,
  abstract = { Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives. },
  month = { Apr },
  pages = { 449--455 },
  number = { 4 },
  volume = { 19 },
  year = { 2012 },
  journal = { Chem. Biol. },
  title = { {S}tructure-based design of supercharged, highly thermoresistant antibodies },
  author = { Miklos and Kluwe and Der and Pai and Sircar and Hughes and Berrondo and Xu and Codrea and Buckley and Calm and Welsh and Warner and Zacharko and Carney and Gray and Georgiou and Kuhlman and Ellington },
}

@article{pmid22558161,
  abstract = { Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ(null) engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ(null) mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D(H)-J(H) pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments. },
  pages = { e35497 },
  number = { 4 },
  volume = { 7 },
  year = { 2012 },
  journal = { PLoS ONE },
  title = { {A}ntibody repertoires in humanized {N}{O}{D}-scid-{I}{L}2{R}γ(null) mice and human {B} cells reveals human-like diversification and tolerance checkpoints in the mouse },
  author = { Ippolito and Hoi and Reddy and Carroll and Ge and Rogosch and Zemlin and Shultz and Ellington and Vandenberg and Georgiou },
}

@article{pmid22670809,
  abstract = { The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). CTD regulation of transcription is mediated by both phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, and thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation. },
  month = { Aug },
  pages = { 1462--1470 },
  number = { 8 },
  volume = { 7 },
  year = { 2012 },
  journal = { ACS Chem. Biol. },
  title = { {S}tructural and kinetic analysis of prolyl-isomerization/phosphorylation cross-talk in the {C}{T}{D} code },
  author = { Zhang and Wang and Chen and Bowman and Luo and Noel and Ellington and Etzkorn and Zhang },
}

@article{pmid22706642,
  abstract = { Epidermal growth factor receptor (EGFR) is a cell surface protein overexpressed in cancerous cells. It is known to be the most common oncogene. EGFR concentration also increases in the serum of cancer patients. The detection of small changes in the concentration of EGFR can be critical for early diagnosis, resulting in better treatment and improved survival rate of cancer patients. This article reports an RNA aptamer based approach to selectively capture EGFR protein and an electrical scheme for its detection. Pairs of gold electrodes with nanometer separation were made through confluence of focused ion beam scratching and electromigration. The aptamer was hybridized to a single stranded DNA molecule, which in turn was immobilized on the SiO(2) surface between the gold nanoelectrodes. The selectivity of the aptamer was demonstrated by using control chips with mutated non-selective aptamer and with no aptamer. Surface functionalization was characterized by optical detection and two orders of magnitude increase in direct current (DC) was measured when selective capture of EGFR occurred. This represents an electronic biosensor for the detection of proteins of interest for medical applications. },
  month = { Jul },
  pages = { 275502 },
  number = { 27 },
  volume = { 23 },
  year = { 2012 },
  journal = { Nanotechnology },
  title = { {E}lectrical detection of cancer biomarker using aptamers with nanogap break-junctions },
  author = { Ilyas and Asghar and Allen and Duhon and Ellington and Iqbal },
}

@article{pmid22729075,
  abstract = { This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format. },
  month = { Aug },
  pages = { 2951--2958 },
  number = { 16 },
  volume = { 12 },
  year = { 2012 },
  journal = { Lab Chip },
  title = { {D}{N}{A} circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform },
  author = { Allen and Arshad and Li and Chen and Ellington },
}

@article{pmid22737599,
  abstract = { Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions. },
  month = { Jun },
  pages = { 221--228 },
  number = { 6 },
  volume = { 1 },
  year = { 2012 },
  journal = { ACS Synth Biol },
  title = { {S}{C}{H}{E}{M}{A}-designed variants of human {A}rginase {I} and {I}{I} reveal sequence elements important to stability and catalysis },
  author = { Romero and Stone and Lamb and Chantranupong and Krause and Miklos and Hughes and Fechtel and Ellington and Arnold and Georgiou },
}

@article{pmid22870858,
  abstract = { The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides. },
  month = { Jul },
  pages = { Unit3.23 },
  volume = { Chapter 3 },
  year = { 2012 },
  journal = { Curr Protoc Mol Biol },
  title = { {D}esign and assembly of large synthetic {D}{N}{A} constructs },
  author = { Miklos and Hughes and Ellington },
}

@article{pmid22870859,
  abstract = { As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment. },
  month = { Jul },
  pages = { Unit3.24 },
  volume = { Chapter 3 },
  year = { 2012 },
  journal = { Curr Protoc Mol Biol },
  title = { {E}nrichment of error-free synthetic {D}{N}{A} sequences by {C}{E}{L} {I} nuclease },
  author = { Hughes and Miklos and Ellington },
}

@article{pmid22891822,
  abstract = { The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic functions (including the replication of nucleic acids) to more competent protein enzymes would complete the journey from an abiotic world to the molecular biology we see today. },
  month = { Dec },
  pages = { 2097--2105 },
  number = { 12 },
  volume = { 45 },
  year = { 2012 },
  journal = { Acc. Chem. Res. },
  title = { {A}biotic self-replication },
  author = { Meyer and Ellefson and Ellington },
}

@article{pmid22894754,
  abstract = { Catalyzed hairpin assembly (CHA) is a robust enzyme-free signal-amplification reaction that has a wide range of potential applications, especially in biosensing. Although most studies of the analytical applications of CHA have focused on the measurement of concentrations of biomolecules, we show here that CHA can also be used to probe the spatial organization of biomolecules such as single-stranded DNA. The basis of such detection is the fact that a DNA structure that brings a toehold and a branch-migration domain into close proximity can catalyze the CHA reaction. We quantitatively studied this phenomenon and applied it to the detection of domain reorganization that occurs during DNA self-assembly processes such as the hybridization chain reaction (HCR). We also show that CHA circuits can be designed to detect certain types of hybridization defects. This principle allowed us to develop a "signal on" assay that can simultaneously respond to multiple types of mutations in a DNA strand in one simple reaction, which is of great interest in genotyping and molecular diagnostics. These findings highlight the potential impacts of DNA circuitry on DNA nanotechnology and provide new tools for further development of these fields. },
  month = { Aug },
  pages = { 13918--13921 },
  number = { 34 },
  volume = { 134 },
  year = { 2012 },
  journal = { J. Am. Chem. Soc. },
  title = { {P}robing spatial organization of {D}{N}{A} strands using enzyme-free hairpin assembly circuits },
  author = { Li and Jiang and Chen and Ellington },
}

@article{pmid22927795,
  abstract = { Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans. },
  pages = { e1001379 },
  number = { 8 },
  volume = { 10 },
  year = { 2012 },
  journal = { PLoS Biol. },
  title = { {E}volutionarily repurposed networks reveal the well-known antifungal drug thiabendazole to be a novel vascular disrupting agent },
  author = { Cha and Byrom and Mead and Ellington and Wallingford and Marcotte },
}

@article{pmid22938015,
  abstract = { Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium. },
  month = { Sep },
  pages = { 15245--15248 },
  number = { 37 },
  volume = { 134 },
  year = { 2012 },
  journal = { J. Am. Chem. Soc. },
  title = { {A}nalysis of {D}{N}{A}-guided self-assembly of microspheres using imaging flow cytometry },
  author = { Tang and Deschner and Allen and Cho and Sermas and Maurer and Ellington and Willson },
}

@article{pmid22947054,
  abstract = { Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~10(9) copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a nonenzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both nonspecific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons. },
  month = { Oct },
  pages = { 8371--8377 },
  number = { 19 },
  volume = { 84 },
  year = { 2012 },
  journal = { Anal. Chem. },
  title = { {A}dapting enzyme-free {D}{N}{A} circuits to the detection of loop-mediated isothermal amplification reactions },
  author = { Li and Chen and Ellington },
}

@article{pmid22983436,
  abstract = { Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices. },
  month = { Nov },
  pages = { 4693--4701 },
  number = { 22 },
  volume = { 12 },
  year = { 2012 },
  journal = { Lab Chip },
  title = { {C}apture, isolation and release of cancer cells with aptamer-functionalized glass bead array },
  author = { Wan and Liu and Allen and Asghar and Mahmood and Tan and Duhon and Kim and Ellington and Iqbal },
}

@article{pmid23018995,
  month = { Oct },
  pages = { 938--939 },
  number = { 10 },
  volume = { 9 },
  year = { 2012 },
  journal = { Nat. Methods },
  title = { {A}ptamers as potential tools for super-resolution microscopy },
  author = { Opazo and Levy and Byrom and Schafer and Geisler and Groemer and Ellington and Rizzoli },
}

@article{pmid23030589,
  abstract = { Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo. },
  month = { Oct },
  pages = { 295--305 },
  number = { 5 },
  volume = { 22 },
  year = { 2012 },
  journal = { Nucleic Acid Ther },
  title = { {A}ptamer-mediated delivery of chemotherapy to pancreatic cancer cells },
  author = { Ray and Cheek and Sharaf and Li and Ellington and Sullenger and Shaw and White },
}

@article{pmid23057741,
  abstract = { Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci--such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis--to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality--and dysfunctionality--of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable. },
  pages = { 89--111 },
  volume = { 28 },
  year = { 2012 },
  journal = { Annu. Rev. Cell Dev. Biol. },
  title = { {D}ynamic reorganization of metabolic enzymes into intracellular bodies },
  author = { O'Connell and Zhao and Ellington and Marcotte },
}

@article{pmid23132138,
  abstract = { DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA) circuit to initiate a hybridization chain reaction (HCR) circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal. },
  pages = { 13211--13220 },
  number = { 11 },
  volume = { 17 },
  year = { 2012 },
  journal = { Molecules },
  title = { {C}oupling two different nucleic acid circuits in an enzyme-free amplifier },
  author = { Jiang and Li and Chen and Ellington },
}

@article{pmid23143151,
  abstract = { We have developed a set of DNA circuits that execute during gel electrophoresis to yield immobile, fluorescent features in the gel. The parallel execution of orthogonal circuits led to the simultaneous production of different fluorescent lines at different positions in the gel. The positions of the lines could be rationally manipulated by changing the mobilities of the reactants. The ability to program at the nanoscale so as to produce patterns at the macroscale is a step towards programmable, synthetic chemical systems for generating defined spatiotemporal patterns. },
  pages = { 13390--13402 },
  number = { 11 },
  volume = { 17 },
  year = { 2012 },
  journal = { Molecules },
  title = { {S}patial control of {D}{N}{A} reaction networks by {D}{N}{A} sequence },
  author = { Allen and Chen and Ellington },
}

@article{pmid23651157,
  abstract = { Recent technological advances have allowed development of increasingly complex systems for in vitro evolution. Here, we describe an in vitro autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture. Compartmentalization of individual templates within a water-in-oil emulsion links genotype and phenotype, allowing evolution. },
  month = { May },
  pages = { 190--196 },
  number = { 5 },
  volume = { 1 },
  year = { 2012 },
  journal = { ACS Synth Biol },
  title = { {A}n in vitro autogene },
  author = { Davidson and Meyer and Ellefson and Levy and Ellington },
}

@article{pmid21299200,
  abstract = { A novel saccharide host containing four boronic acid recognition units on a single DNA duplex terminus was constructed. This construct allowed boronic acid sugar recognition in the context of double-stranded DNA to be established while highlighting the benefits of multivalency. Following the solid-phase synthesis of a bis-boronic acid tag, two end-functionalized oligonucleotides with complementary sequences were functionalized through amide ligation. By annealing the boronic acid-DNA conjugates, a tetra-boronic acid DNA duplex was assembled. The saccharide binding ability of this tetra-boronic acid host was revealed through cellulose paper chromatography in the absence and presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNA conjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selected monosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognition sites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems to the realm of macromolecules. },
  month = { Mar },
  pages = { 388--396 },
  number = { 3 },
  volume = { 22 },
  year = { 2011 },
  journal = { Bioconjug. Chem. },
  title = { {C}hemical functionalization of oligodeoxynucleotides with multiple boronic acids for the polyvalent binding of saccharides },
  author = { Hargrove and Ellington and Anslyn and Sessler },
}

@article{pmid21338059,
  abstract = { In order to automate the optimization of complex biochemical and molecular biology reactions, we developed a sequential injection analysis (SIA) device and combined this with a design of experiment (DOE) algorithm. This combination of hardware and software automatically explores the parameter space of the reaction and provides continuous feedback for optimizing reaction conditions. As an example, we optimized the endonuclease digest of a fluorogenic substrate and showed that the optimized reaction conditions also applied to the digest of the substrate outside of the device and to the digest of a plasmid. The sequential technique quickly arrived at optimized reaction conditions with less reagent use than a batch process (such as a fluid handling robot exploring multiple reaction conditions in parallel) would have. The device and method should now be amenable to much more complex molecular biology reactions whose variable spaces are correspondingly larger. },
  month = { Mar },
  pages = { 2194--2200 },
  number = { 6 },
  volume = { 83 },
  year = { 2011 },
  journal = { Anal. Chem. },
  title = { {S}equential injection analysis for optimization of molecular biology reactions },
  author = { Allen and Ellington },
}

@article{pmid21601682,
  abstract = { DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided. },
  pages = { 277--309 },
  volume = { 498 },
  year = { 2011 },
  journal = { Meth. Enzymol. },
  title = { {G}ene synthesis: methods and applications },
  author = { Hughes and Miklos and Ellington },
}

@article{pmid21654670,
  month = { Jun },
  pages = { 502--503 },
  number = { 6 },
  volume = { 29 },
  year = { 2011 },
  journal = { Nat. Biotechnol. },
  title = { M}olecular evolution picks up the {P}{A}{C}{E },
  author = { Meyer and Ellington },
}

@article{pmid21687663,
  abstract = { Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K(d) = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents. },
  pages = { e20299 },
  number = { 6 },
  volume = { 6 },
  year = { 2011 },
  journal = { PLoS ONE },
  title = { {I}nhibition of cell proliferation by an anti-{E}{G}{F}{R} aptamer },
  author = { Li and Nguyen and Byrom and Ellington },
}

@article{pmid21693555,
  abstract = { Signal amplification is a key component of molecular detection. Enzyme-free signal amplification is especially appealing for the development of low-cost, point-of-care diagnostics. It has been previously shown that enzyme-free DNA circuits with signal-amplification capacity can be designed using a mechanism called 'catalyzed hairpin assembly'. However, it is unclear whether the efficiency and modularity of such circuits is suitable for multiple analytical applications. We have therefore designed and characterized a simplified DNA circuit based on catalyzed hairpin assembly, and applied it to multiple different analytical formats, including fluorescent, colorimetric, and electrochemical and signaling. By optimizing the design of previous hairpin-based catalytic assemblies we found that our circuit has almost zero background and a high catalytic efficiency, with a k(cat) value above 1 min(-1). The inherent modularity of the circuit allowed us to readily adapt our circuit to detect both RNA and small molecule analytes. Overall, these data demonstrate that catalyzed hairpin assembly is suitable for analyte detection and signal amplification in a 'plug-and-play' fashion. },
  month = { Sep },
  pages = { e110 },
  number = { 16 },
  volume = { 39 },
  year = { 2011 },
  journal = { Nucleic Acids Res. },
  title = { {R}ational, modular adaptation of enzyme-free {D}{N}{A} circuits to multiple detection methods },
  author = { Li and Ellington and Chen },
}

@article{pmid21938060,
  month = { Sep },
  pages = { 413--414 },
  number = { 7365 },
  volume = { 477 },
  year = { 2011 },
  journal = { Nature },
  title = { {S}ynthetic biology: a yeast for all reasons },
  author = { Enyeart and Ellington },
}

@article{pmid22019591,
  abstract = { The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins. },
  month = { Dec },
  pages = { 356--369 },
  number = { 3 },
  volume = { 414 },
  year = { 2011 },
  journal = { J. Mol. Biol. },
  title = { {S}tructure of the {E}scherichia coli phosphonate binding protein {P}hn{D} and rationally optimized phosphonate biosensors },
  author = { Alicea and Marvin and Miklos and Ellington and Looger and Schreiter },
}

@article{pmid19908395,
  abstract = { The ability to build and control complex biological systems is greatly enhanced by the generation of related parts with varying strengths. In this way, various parts can be strung together and the connectivity and expression levels can be matched for the desired system performance. Engineered gene circuits, both in vivo and in vitro, often utilize the T7 RNA polymerase in tandem with the T7 promoter for transcription. In this work, we describe the selection of T7 promoter variants of varying strength by emulsifying in vitro transcription with subsequent fluorescence activated cell sorting (FACS) to enrich for active promoters. Such variant promoters should be of use to synthetic biologists for both in vivo and in vitro applications. },
  pages = { 433--443 },
  year = { 2010 },
  journal = { Pac Symp Biocomput },
  title = { {E}mulsion based selection of {T}7 promoters of varying activity },
  author = { Davidson and VAN Blarcom and Levy and Ellington },
}

@article{pmid19947589,
  abstract = { We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor. },
  month = { Jan },
  pages = { 197--202 },
  number = { 1 },
  volume = { 82 },
  year = { 2010 },
  journal = { Anal. Chem. },
  title = { {D}iffractometric detection of proteins using microbead-based rolling circle amplification },
  author = { Lee and Icoz and Roberts and Ellington and Savran },
}

@article{pmid20066302,
  abstract = { A newly selected anti-receptor (anti-EGFR) aptamer was conjugated to gold nanoparticles via a facile hybridization method and was found to specifically and quantitatively direct the delivery of gold nanoparticles to cells expressing EGFR through receptor-mediated endocytosis. },
  month = { Jan },
  pages = { 392--394 },
  number = { 3 },
  volume = { 46 },
  year = { 2010 },
  journal = { Chem. Commun. (Camb.) },
  title = { {D}irected evolution of gold nanoparticle delivery to cells },
  author = { Li and Larson and Nguyen and Sokolov and Ellington },
}

@article{pmid20129797,
  abstract = { Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5' P-O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for approximately 25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5' P-O bonds began to populate the product ion mass spectra as well as higher abundances of [a - Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a - Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H](+), [A + H](+), and [C + H](+), which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID. },
  month = { Apr },
  pages = { 580--591 },
  number = { 4 },
  volume = { 21 },
  year = { 2010 },
  journal = { J. Am. Soc. Mass Spectrom. },
  title = { {I}nfrared multiphoton dissociation of small-interfering {R}{N}{A} anions and cations },
  author = { Gardner and Li and Ellington and Brodbelt },
}

@article{pmid20198660,
  abstract = { We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions. },
  month = { Jun },
  pages = { 347--357 },
  number = { 3 },
  volume = { 106 },
  year = { 2010 },
  journal = { Biotechnol. Bioeng. },
  title = { {R}apid construction and characterization of synthetic antibody libraries without {D}{N}{A} amplification },
  author = { Ge and Mazor and Hunicke-Smith and Ellington and Georgiou },
}

@article{pmid20201028,
  abstract = { This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. },
  month = { Mar },
  pages = { 1--27 },
  volume = { Chapter 9 },
  year = { 2010 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization },
  author = { Hall and Arshad and Seo and Bowman and Corley and Jhaveri and Ellington },
}

@article{pmid20300533,
  abstract = { Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators. },
  pages = { e9726 },
  number = { 3 },
  volume = { 5 },
  year = { 2010 },
  journal = { PLoS ONE },
  title = { {A}ptamer antagonists of myelin-derived inhibitors promote axon growth },
  author = { Wang and Khaing and Li and Hall and Schmidt and Ellington },
}

@article{pmid20501662,
  abstract = { The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related gamma-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators. },
  month = { Jul },
  pages = { 22840--22845 },
  number = { 30 },
  volume = { 285 },
  year = { 2010 },
  journal = { J. Biol. Chem. },
  title = { {I}dentification of novel specific allosteric modulators of the glycine receptor using phage display },
  author = { Tipps and Lawshe and Ellington and Mihic },
}

@article{pmid20538451,
  abstract = { Nucleic acid-based nanotechnology has always been perceived as novel, but has begun to move from theoretical demonstrations to practical applications. In particular, the large address spaces available to nucleic acids can be exploited to encode algorithms and/or act as circuits and thereby process molecular information. In this review we not only revisit several milestones in the field of nucleic acid-based computation, but also highlight how the prospects for nucleic acid computation go beyond just a large address space. Functional nucleic acid elements (aptamers, ribozymes, and deoxyribozymes) can serve as inputs and outputs to the environment, and can act as logical elements. Into the future, the chemical dynamics of nucleic acids may prove as useful as hybridization for computation. },
  month = { Aug },
  pages = { 392--400 },
  number = { 4 },
  volume = { 21 },
  year = { 2010 },
  journal = { Curr. Opin. Biotechnol. },
  title = { {S}haping up nucleic acid computation },
  author = { Chen and Ellington },
}

@article{pmid20571084,
  abstract = { While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity. },
  month = { Oct },
  pages = { 6813--6830 },
  number = { 19 },
  volume = { 38 },
  year = { 2010 },
  journal = { Nucleic Acids Res. },
  title = { R}ational design of an orthogonal tryptophanyl nonsense suppressor t{R}{N}{A },
  author = { Hughes and Ellington },
}

@article{pmid20802495,
  abstract = { Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities. },
  month = { Sep },
  pages = { 965--969 },
  number = { 9 },
  volume = { 28 },
  year = { 2010 },
  journal = { Nat. Biotechnol. },
  title = { {M}onoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells },
  author = { Reddy and Ge and Miklos and Hughes and Kang and Hoi and Chrysostomou and Hunicke-Smith and Iverson and Tucker and Ellington and Georgiou },
}

@article{pmid20947753,
  month = { Oct },
  pages = { 330--331 },
  number = { 6002 },
  volume = { 330 },
  year = { 2010 },
  journal = { Science },
  title = { E}volution. {R}{N}{A} {G}{P}{S },
  author = { Kluwe and Ellington },
}

@article{pmid21060734,
  abstract = { Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 10(8) bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 10(1) to 10(5) cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial "lobster traps") defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (10(12) cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations. },
  pages = {   },
  number = { 4 },
  volume = { 1 },
  year = { 2010 },
  journal = { MBio },
  title = { {P}robing prokaryotic social behaviors with bacterial "lobster traps" },
  author = { Connell and Wessel and Parsek and Ellington and Whiteley and Shear },
}

@article{pmid21062984,
  abstract = { Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment. },
  month = { Nov },
  pages = { 9371--9380 },
  number = { 22 },
  volume = { 70 },
  year = { 2010 },
  journal = { Cancer Res. },
  title = { {S}urface-immobilized aptamers for cancer cell isolation and microscopic cytology },
  author = { Wan and Kim and Li and Cho and Bachoo and Ellington and Iqbal },
}

@article{pmid23850755,
  abstract = { In this paper, we present a fully integrated biosensor 10 × 10 array in a standard complementary metal-oxide semiconducor process, which takes advantage of electrochemical impedance spectroscopy (EIS). We also show that this system is able to detect various biological analytes, such as DNA and proteins, in real time and without the need for molecular labels. In each pixel of this array, we implement a biocompatible Au electrode transducer and embedded sensor circuitry which takes advantage of the coherent detector to measure the impedance of the associated electrode-electrolyte interface. This chip is capable of concurrently measuring admittance values as small as 10(-8) Ω(-1) within the array with the detection dynamic range of more than 90 dB in the frequency range of 10 Hz-50 MHz. },
  month = { Dec },
  pages = { 379--390 },
  number = { 6 },
  volume = { 4 },
  year = { 2010 },
  journal = { IEEE Trans Biomed Circuits Syst },
  title = { {A} {C}{M}{O}{S} {E}lectrochemical {I}mpedance {S}pectroscopy ({E}{I}{S}) {B}iosensor {A}rray },
  author = { Manickam and Chevalier and McDermott and Ellington and Hassibi },
}

@article{pmid18775793,
  abstract = { In vitro selection experiments show first and foremost that it is possible that functional nucleic acids can arise from random sequence libraries. Indeed, even simple sequence and structural motifs can prove to be robust binding species and catalysts, indicating that it may have been possible to transition from even the earliest self-replicators to a nascent, RNA-catalyzed metabolism. Because of the diversity of aptamers and ribozymes that can be selected, it is possible to construct a 'fossil record' of the evolution of the RNA world, with in vitro selected catalysts filling in as doppelgangers for molecules long gone. In this way a plausible pathway from simple oligonucleotide replicators to genomic polymerases can be imagined, as can a pathway from basal ribozyme activities to the ribosome. Most importantly, though, in vitro selection experiments can give a true and quantitative idea of the likelihood that these scenarios could have played out in the RNA world. Simple binding species and catalysts could have evolved into other structures and functions. As replicating sequences grew longer, new, more complex functions or faster catalytic activities could have been accessed. Some activities may have been isolated in sequence space, but others could have been approached along large, interconnected neutral networks. As the number, type, and length of ribozymes increased, RNA genomes would have evolved and eventually there would have been no area in a fitness landscape that would have been inaccessible. Self-replication would have inexorably led to life. },
  month = { Feb },
  pages = { 254--265 },
  number = { 2 },
  volume = { 41 },
  year = { 2009 },
  journal = { Int. J. Biochem. Cell Biol. },
  title = { E}volutionary origins and directed evolution of {R}{N}{A },
  author = { Ellington and Chen and Robertson and Syrett },
}

@article{pmid19053807,
  abstract = { Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip. },
  month = { Jan },
  pages = { 6--19 },
  number = { 1 },
  volume = { 8 },
  year = { 2009 },
  journal = { J. Proteome Res. },
  title = { {S}ystematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast },
  author = { Narayanaswamy and Moradi and Niu and Hart and Davis and McGary and Ellington and Marcotte },
}

@article{pmid19159107,
  abstract = { The detection and typing of tumor cells based on differentially or similarly expressed antigens (biomarkers) have proven to be increasingly important for the diagnosis and treatment of various cancers. Sensitive techniques for the detection of cell surface antigens are therefore crucial for the early and accurate detection of cancer. Although techniques such as ELISA and tissue staining have proven their worth, these techniques often either require substantial amounts of starting material or are prone to high background and false negatives. The proximity ligation assay (PLA) has proven to be an exquisitely sensitive technique with very low background. Two probes that bind adjacent to one another on a protein target can be ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR. We have now adapted PLA to cell surface protein targets using modified RNA aptamers, and have shown that aptamer-based cell surface PLA can successfully detect and differentiate between cells that differentially express a tumor antigen, the prostate specific membrane antigen (PSMA). },
  pages = { 385--398 },
  volume = { 504 },
  year = { 2009 },
  journal = { Methods Mol. Biol. },
  title = { {U}sing {R}{N}{A} aptamers and the proximity ligation assay for the detection of cell surface antigens },
  author = { Pai and Ellington },
}

@article{pmid19177559,
  abstract = { Disulfide bonds play a critical role in the stabilization of the immunoglobulin beta-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues. },
  month = { Feb },
  pages = { 259--267 },
  number = { 2 },
  volume = { 18 },
  year = { 2009 },
  journal = { Protein Sci. },
  title = { {E}ngineering antibody fragments to fold in the absence of disulfide bonds },
  author = { Seo and Jeong and Leysath and Ellington and Iverson and Georgiou },
}

@article{pmid19271740,
  abstract = { A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome. },
  month = { May },
  pages = { 2438--2448 },
  number = { 5 },
  volume = { 8 },
  year = { 2009 },
  journal = { J. Proteome Res. },
  title = { {T}echnical and biological issues relevant to cell typing with aptamers },
  author = { Li and Ebright and Stovall and Chen and Nguyen and Singh and Syrett and Ellington },
}

@article{pmid19295523,
  month = { Apr },
  pages = { 200--201 },
  number = { 4 },
  volume = { 5 },
  year = { 2009 },
  journal = { Nat. Chem. Biol. },
  title = { {B}ack to the future of nucleic acid self-amplification },
  author = { Ellington },
}

@article{pmid19377997,
  abstract = { Random sequence nucleic acid pools can be used in a variety of applications, including the selection of functional nucleic acids such as protein binding sites, aptamers, and ribozymes. while the design, synthesis, and purification of pools is relatively straightforward, keeping track of the size and complexity of a nucleic acid pool can sometimes task even an experienced researcher. The following protocol takes the reader through the steps necessary for the preparation of a pool of known complexity. },
  pages = { 3--18 },
  volume = { 535 },
  year = { 2009 },
  journal = { Methods Mol. Biol. },
  title = { {N}ucleic acid pool preparation and characterization },
  author = { Piasecki and Hall and Ellington },
}

@article{pmid19431189,
  abstract = { Aptamers have been utilized as biosensors because they can be readily adapted to sensor platforms and signal transduction schemes through both rational design and selection. One highly generalizable scheme for the generation of the so-called aptamer beacons involves denaturing the aptamer with antisense oligonucleotides. For example, rational design methods have been utilized to adapt anti-thrombin aptamers to function as biosensors by hybridizing an antisense oligonucleotide containing a quencher to the aptamer containing a fluorescent label. In the presence of thrombin, the binding equilibrium is shifted, the antisense oligonucleotide dissociates, and the beacon lights up. By changing the affinity of the antisense oligonucleotide for the aptamer beacon, it has proven possible to change the extent of activation of the beacon. More importantly, modulating interactions between the antisense oligonucleotide and the aptamer strongly influences the kinetics of activation. Comparisons across multiple, designed aptamer beacons indicate that there is a strong inverse correlation between the thermodynamics of hybridization and the speed of activation, a finding that should prove to be generally useful in the design of future biosensors. By pre-organizing the thrombin-binding quadruplex within the aptamer the speed of response can be greatly increased. By integrating these various interactions, we were ultimately able to design aptamer beacons that were activated by threefold within 1 min of the addition of thrombin. },
  month = { Aug },
  pages = { 1049--1059 },
  number = { 6 },
  volume = { 103 },
  year = { 2009 },
  journal = { Biotechnol. Bioeng. },
  title = { {K}inetic optimization of a protein-responsive aptamer beacon },
  author = { Hall and Cater and Levy and Ellington },
}

@article{pmid19474083,
  abstract = { The power of electronic computation is due in part to the development of modular gate structures that can be coupled to carry out sophisticated logical operations and whose performance can be readily modelled. However, the equivalences between electronic and biochemical operations are far from obvious. In order to help cross between these disciplines, we develop an analogy between complementary metal oxide semiconductor and transcriptional logic gates. We surmise that these transcriptional logic gates might prove to be useful in amorphous computations and model the abilities of immobilized gates to form patterns. Finally, to begin to implement these computations, we design unique hairpin transcriptional gates and then characterize these gates in a binary latch similar to that already demonstrated by Kim et al. (Kim, White & Winfree 2006 Mol. Syst. Biol. 2, 68 (doi:10.1038/msb4100099)). The hairpin transcriptional gates are uniquely suited to the design of a complementary NAND gate that can serve as an underlying basis of molecular computing that can output matter rather than electronic information. },
  month = { Aug },
  pages = { S523--533 },
  volume = { 6 Suppl 4 },
  year = { 2009 },
  journal = { J R Soc Interface },
  title = { {M}odelling amorphous computations with transcription networks },
  author = { Simpson and Tsai and Nguyen and Chen and Ellington },
}

@article{pmid19502427,
  abstract = { Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When approximately 800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution. },
  month = { Jun },
  pages = { 10147--10152 },
  number = { 25 },
  volume = { 106 },
  year = { 2009 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {W}idespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation },
  author = { Narayanaswamy and Levy and Tsechansky and Stovall and O'Connell and Mirrielees and Ellington and Marcotte },
}

@article{pmid19563759,
  abstract = { Edge detection is a signal processing algorithm common in artificial intelligence and image recognition programs. We have constructed a genetically encoded edge detection algorithm that programs an isogenic community of E. coli to sense an image of light, communicate to identify the light-dark edges, and visually present the result of the computation. The algorithm is implemented using multiple genetic circuits. An engineered light sensor enables cells to distinguish between light and dark regions. In the dark, cells produce a diffusible chemical signal that diffuses into light regions. Genetic logic gates are used so that only cells that sense light and the diffusible signal produce a positive output. A mathematical model constructed from first principles and parameterized with experimental measurements of the component circuits predicts the performance of the complete program. Quantitatively accurate models will facilitate the engineering of more complex biological behaviors and inform bottom-up studies of natural genetic regulatory networks. },
  month = { Jun },
  pages = { 1272--1281 },
  number = { 7 },
  volume = { 137 },
  year = { 2009 },
  journal = { Cell },
  title = { {A} synthetic genetic edge detection program },
  author = { Tabor and Salis and Simpson and Chevalier and Levskaya and Marcotte and Voigt and Ellington },
}

@article{pmid19575478,
  abstract = { This unit describes a protocol for the directed evolution of proteins utilizing in vitro compartmentalization. This method uses a large number of independent in vitro transcription and translation (IVTT) reactions in water droplets suspended in an oil emulsion to enable selection of proteins that bind a target molecule. Protein variants that bind the target also bind to and allow recovery of the genes that encoded them. This protocol serves as a basis for carrying out selections in emulsions, and can potentially be modified to select for other functionalities, including catalysis. This selection method is advantageous compared to alternative selection protocols due to the ability to screen through very large-size libraries and the ability to express and screen or select for functions that would otherwise be toxic or inaccessible to in vivo selections and screens. },
  month = { Jul },
  pages = { Unit 24.6 },
  volume = { Chapter 24 },
  year = { 2009 },
  journal = { Curr Protoc Mol Biol },
  title = { {D}irected evolution of proteins in vitro using compartmentalization in emulsions },
  author = { Davidson and Dlugosz and Levy and Ellington },
}

@article{pmid19735583,
  abstract = { Because of the increasing recognition of the importance of non-coding RNAs in gene regulation, there is considerable interest in identifying RNA motifs in genomic data. In a recent report in BMC Genomics, Breaker and colleagues describe a new algorithm for identifying functional noncoding RNAs in metagenomic sequences of marine organisms, a strategy that may be particularly effective for discovering new and unique riboswitches. },
  pages = { 72 },
  number = { 8 },
  volume = { 8 },
  year = { 2009 },
  journal = { J. Biol. },
  title = { {M}otifs from the deep },
  author = { Hwang and Codrea and Ellington },
}

@article{pmid19776159,
  abstract = { Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments. },
  month = { Nov },
  pages = { 2035--2045 },
  number = { 11 },
  volume = { 15 },
  year = { 2009 },
  journal = { RNA },
  title = { {D}irect selection for ribozyme cleavage activity in cells },
  author = { Chen and Denison and Levy and Ellington },
}

@article{pmid19816932,
  abstract = { Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions. },
  month = { Oct },
  pages = { Unit 24.2 },
  volume = { Chapter 24 },
  year = { 2009 },
  journal = { Curr Protoc Mol Biol },
  title = { {D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection },
  author = { Hall and Micheletti and Satya and Ogle and Pollard and Ellington },
}

@article{pmid19816933,
  abstract = { This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. },
  month = { Oct },
  pages = { Unit 24.3 },
  volume = { Chapter 24 },
  year = { 2009 },
  journal = { Curr Protoc Mol Biol },
  title = { {I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization },
  author = { Hall and Arshad and Seo and Bowman and Corley and Jhaveri and Ellington },
}

@article{pmid20013786,
  abstract = { Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions. },
  month = { Dec },
  pages = { Unit 9.2 },
  volume = { Chapter 9 },
  year = { 2009 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection },
  author = { Hall and Micheletti and Satya and Ogle and Pollard and Ellington },
}

@article{pmid20041206,
  abstract = { Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications. },
  month = { Dec },
  pages = { e1000620 },
  number = { 12 },
  volume = { 5 },
  year = { 2009 },
  journal = { PLoS Comput. Biol. },
  title = { {D}esign principles for ligand-sensing, conformation-switching ribozymes },
  author = { Chen and Ellington },
}

@article{pmid20636061,
  abstract = { Aptamers are ligand-binding nucleic acids whose affinities and selectivities can rival those of antibodies. They have been adapted to analytical applications not only as alternatives to antibodies, but as unique reagents in their own right. In particular, aptamers can be readily site-specifically modified during chemical or enzymatic synthesis to incorporate particular reporters, linkers, or other moieties. Also, aptamer secondary structures can be engineered to undergo analyte-dependent conformational changes, which, in concert with the ability to specifically place chemical agents, opens up a wealth of possible signal transduction schemas, irrespective of whether the detection modality is optical, electrochemical, or mass based. Finally, because aptamers are nucleic acids, they are readily adapted to sequence- (and hence signal-) amplification methods. However, application of aptamers without a basic knowledge of their biochemistry or technical requirements can cause serious analytical difficulties. },
  pages = { 241--264 },
  volume = { 2 },
  year = { 2009 },
  journal = { Annu Rev Anal Chem (Palo Alto Calif) },
  title = { {A}pplications of aptamers as sensors },
  author = { Cho and Lee and Ellington },
}

@article{pmid18049815,
  abstract = { In order to generate nucleic acid biosensors that could undergo a reversible conformation change in the presence of the metal zinc, a random sequence pool of single-stranded DNA was immobilized on an oligonucleotide affinity column. In the presence of zinc, those species that underwent a conformational change were released from the column, collected, and amplified. A series of negative and positive selections refined the metal specificity of the selected aptamer beacons. Since the aptamer beacons contained a fluorophore, while the bound oligonucleotide contained a quencher, zinc binding also resulted in an increase in fluorescence. One of the selected beacons, Zn-6m2, bound zinc in the low micromolar range, gave a dose-dependent fluorescence signal, and showed an approximately sixfold increase in fluorescence on zinc binding. While some cross-reactivity with cadmium was observed, it should nonetheless prove possible to use the novel selection method to generate and tune the specificity of a variety of reversible metal biosensors. Such biosensors could potentially be used for continuous monitoring of metals in environmental samples. },
  month = { Feb },
  pages = { 1067--1075 },
  number = { 4 },
  volume = { 390 },
  year = { 2008 },
  journal = { Anal Bioanal Chem },
  title = { {S}election of fluorescent aptamer beacons that light up in the presence of zinc },
  author = { Rajendran and Ellington },
}

@article{pmid18398956,
  month = { Mar },
  pages = { R184--185 },
  number = { 5 },
  volume = { 18 },
  year = { 2008 },
  journal = { Curr. Biol. },
  title = { {Q} &{A}. {A}ndrew {D}. {E}llington },
  author = { Ellington },
}

@article{pmid18507505,
  month = { May },
  pages = { e132 },
  number = { 5 },
  volume = { 6 },
  year = { 2008 },
  journal = { PLoS Biol. },
  title = { {M}an versus machine versus ribozyme },
  author = { Ellington },
}

@article{pmid18541130,
  abstract = { We have developed a novel method that uses conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte and a different secondary structure in the presence of a protein such as thrombin or platelet-derived growth factor (PDGF). The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (because no signal is produced by background binding) and the proximity ligation assay (PLA) (because only one epitope, rather than two epitopes, on a protein surface must be bound). },
  month = { Sep },
  pages = { 164--173 },
  number = { 2 },
  volume = { 380 },
  year = { 2008 },
  journal = { Anal. Biochem. },
  title = { {R}eal-time {P}{C}{R} detection of protein analytes with conformation-switching aptamers },
  author = { Yang and Ellington },
}

@article{pmid18563250,
  abstract = { Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise. },
  month = { Jul },
  pages = { 754--761 },
  number = { 7 },
  volume = { 4 },
  year = { 2008 },
  journal = { Mol Biosyst },
  title = { {E}ngineering stochasticity in gene expression },
  author = { Tabor and Bayer and Simpson and Levy and Ellington },
}

@article{pmid18594898,
  abstract = { Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust. },
  month = { Jul },
  pages = { 95--102 },
  number = { 1 },
  volume = { 67 },
  year = { 2008 },
  journal = { J. Mol. Evol. },
  title = { {B}ioinformatic analysis of the contribution of primer sequences to aptamer structures },
  author = { Cowperthwaite and Ellington },
}

@article{pmid18804035,
  abstract = { We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples. },
  month = { Sep },
  pages = { 979--989 },
  number = { 9 },
  volume = { 15 },
  year = { 2008 },
  journal = { Chem. Biol. },
  title = { {D}irected evolution of streptavidin variants using in vitro compartmentalization },
  author = { Levy and Ellington },
}

@article{pmid18948292,
  abstract = { Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance. },
  month = { Dec },
  pages = { 6739--6751 },
  number = { 21 },
  volume = { 36 },
  year = { 2008 },
  journal = { Nucleic Acids Res. },
  title = { {A}ptamers that recognize drug-resistant {H}{I}{V}-1 reverse transcriptase },
  author = { Li and Wang and Pothukuchy and Syrett and Husain and Gopalakrisha and Kosaraju and Ellington },
}

@article{pmid19008359,
  month = { Nov },
  pages = { 17593--17594 },
  number = { 46 },
  volume = { 105 },
  year = { 2008 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {R}olling out {D}{N}{A} nanostructures in vivo },
  author = { Paukstelis and Ellington },
}

@article{pmid19009652,
  abstract = { To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated.\\ To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].\\ Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells.\\ We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication. },
  month = { Nov },
  pages = { 6360--6365 },
  number = { 41 },
  volume = { 14 },
  year = { 2008 },
  journal = { World J. Gastroenterol. },
  title = { {P}rotective effects of anti-ricin {A}-chain {R}{N}{A} aptamer against ricin toxicity },
  author = { Fan and Wu and Martiniuk and Hale and Ellington and Tchou-Wong },
}

@article{pmid23496781,
  abstract = { Aptamers are selected nucleic acids that bind their targets with affinities and specificities that are often comparable to those of monoclonal antibodies.\\ Although aptamers have been adapted to a wide variety of assay formats, the fact that they are nucleic acids makes them uniquely useful for adaptation to amplification assays. Aptamer-based amplification assays have not previously been reviewed separately.\\ Aptamers can be used as simple binding reagents and then detected by methods such as the polymerase chain reaction and the rolling circle amplification assay. In addition, though, aptamers can undergo programmed, ligand-dependent conformational changes, or can form unique quarternary structures that lead to amplification.\\ Analytical assays that involve aptamers and nuleic acid amplification technologies can be used to detect sensitively target proteins, often in the nanomolar or picomolar range. However, in many cases there are no obvious advantages to amplification assays relative to other assay formats. In all likelihood all formats are limited by the dissociation constants of the aptamers themselves. The one exception to this is the proximity ligation assay, where sequence amplification allows the detection of extremely small quantities of ligands relative to background. },
  month = { Dec },
  pages = { 1333--1346 },
  number = { 12 },
  volume = { 2 },
  year = { 2008 },
  journal = { Expert Opin Med Diagn },
  title = { {A}ptamer amplification: divide and signal },
  author = { Pai and Roberts and Ellington },
}

@article{pmid19121689,
  abstract = { Improved diagnostic reagents would be of considerable benefit in enhancing the specificity and sensitivity of rapid assays for Burkholderia pseudomallei, the causative agent of melioidosis. The purpose of this work is to develop aptamers, high affinity RNA-based molecular recognition molecules, which could be used as reagents for identification of the whole organism in assays of biological samples. Data are presented demonstrating the purification of recombinant B. pseudomallei secreted or surface-exposed macromolecules, which have been expressed in Escherichia coli, and the initial stages of aptamer generation using these recombinant proteins. Future studies will focus upon the expansion of this methodology to include other target macromolecules located on or near the outer membrane of this organism. },
  month = { Dec },
  pages = { S55--57 },
  volume = { 102 Suppl 1 },
  year = { 2008 },
  journal = { Trans. R. Soc. Trop. Med. Hyg. },
  title = { {D}evelopment of aptamers specific for potential diagnostic targets in {B}urkholderia pseudomallei },
  author = { Gnanam and Hall and Shen and Piasecki and Vernados and Galyov and Smither and Kitto and Titball and Ellington and Brown },
}

@article{pmid16713750,
  abstract = { The efficiency of electron transduction by Shewanella oneidensis MR-1 was investigated both in batch culture and in a dual-chambered electrochemical cell. Aerobically grown bacteria were inoculated into an insoluble FeOOH suspension in an anaerobic environment. As the bacteria reduced Fe(III) to Fe(II) there was a visible color change from red to bluish black; this simple color change assay proved to be a robust method for determining the electrochemical activity of S. oneidensis MR-1. In an effort to improve electricity production by S. oneidensis MR-1, the performance of the electrochemical cell with lactate as a fuel was first optimized with respect to both poised potential and fuel concentration. Ultimately, electricity production proved to be proportional to both the poised potential and to fuel concentration. In particular, higher poised potentials increased charge production. Finally, we attempted to optimize the bacteria themselves for the efficient transduction of reduced carbon sources into electricity. The batch culture underwent a series of serial dilutions; after the 4th dilution the microbe population exhibited a 30% increase in charge production. We are further exploring whether this increase was due to metabolic adaptations or to genetic mutations, and examining additional ways to evolve electrogenic organisms. },
  month = { Jan },
  pages = { 165--172 },
  number = { 1 },
  volume = { 70 },
  year = { 2007 },
  journal = { Bioelectrochemistry },
  title = { {O}ptimization of the biological component of a bioelectrochemical cell },
  author = { Cho and Ellington },
}

@article{pmid16996258,
  abstract = { Aptamers have been shown to undergo ligand-dependent conformational changes, and can be joined to ribozymes to create allosteric ribozymes (aptazymes). An anti-flavin (FMN) aptamer joined to the hammerhead ribozyme yielded an aptazyme that underwent small, FMN-dependent displacements in the helix that joined the aptamer and ribozyme. This 'slip structure' model in which alternative sets of base-pairs are formed in the absence and presence of ligand proved amenable to energetic and computational modeling. Initial successes in modeling the activities of known aptazymes led to the in silico selection of new ligand-dependent aptazymes from virtual pools that contained millions of members. Those aptazymes that were predicted to best fit the slip structure model were synthesized and assayed, and the best-designed aptazyme was activated 60-fold by FMN. The slip structure model proved to be generalizable, and could be applied with equal facility to computationally generate aptazymes that proved to be experimentally activated by other ligands (theophylline) or that contained other catalytic cores (hairpin ribozyme). Moreover, the slip structure model could be applied to the prediction of a ligand-dependent aptamer beacon biosensor in which the addition of the protein vascular endothelial growth factor (VegF) led to a 10-fold increase in fluorescent signal. },
  month = { Apr },
  pages = { 1939--1947 },
  number = { 9-10 },
  volume = { 22 },
  year = { 2007 },
  journal = { Biosens Bioelectron },
  title = { {C}omputational selection of nucleic acid biosensors via a slip structure model },
  author = { Hall and Hesselberth and Ellington },
}

@article{pmid17041257,
  abstract = { The incorporation of amino acid analogs is becoming increasingly useful. Site-specific incorporation of unnatural amino acids allows the application of chemical biology to protein-specific investigations and applications. However, the global incorporation of unnatural amino acids allows for tests of proteomic and genetic code hypotheses. For example, the adaptation of organisms to unnatural amino acids may lead to new genetic codes. To understand and quantify changes from such perturbations, an understanding is required of the microbiological and proteomic responses to the incorporation of unnatural amino acids. Here we describe protocols to characterize the effects of such proteome-wide perturbations. },
  pages = { 23--34 },
  volume = { 352 },
  year = { 2007 },
  journal = { Methods Mol. Biol. },
  title = { {G}lobal incorporation of unnatural amino acids in {E}scherichia coli },
  author = { Bacher and Ellington },
}

@article{pmid17173026,
  abstract = { Natural and engineered RNA 'parts' can perform a variety of functions, including hybridizing to targets, binding ligands and undergoing programmed conformational changes, and catalyzing reactions. These RNA parts can in turn be assembled into synthetic genetic circuits that regulate gene expression by acting either in cis or in trans on mRNAs. As more parts are discovered and engineered, it should be increasingly possible to create synthetic RNA circuits that are able to carry out complex logical operations in cells, either superimposed on or autonomous to extant gene regulation. },
  month = { Jan },
  pages = { 23--28 },
  number = { 1 },
  volume = { 3 },
  year = { 2007 },
  journal = { Nat. Chem. Biol. },
  title = { {S}ynthetic {R}{N}{A} circuits },
  author = { Davidson and Ellington },
}

@article{pmid17378540,
  abstract = { Real-time nucleic acid amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to protein or other analytes. To facilitate the development of real-time rolling circle amplification (RCA) for protein targets, we have developed a novel type of conformation-switching aptamer that can be circularized upon interaction with its protein target, the platelet-derived growth factor (PDGF). Using the structure-switching aptamer, real-time RCA can be used to specifically quantitate PDGF down to the low-nanomolar range (limit of detection, 0.4 nM), even against a background of cellular lysate. The aptamer can also be adapted to RCA on surfaces, although quantitation proved to be more difficult. One of the great advantages of the method described herein is that it can be immediately adapted to almost any aptamer and does not require two or more affinity reagents as do sandwich or proximity assays. },
  month = { May },
  pages = { 3320--3329 },
  number = { 9 },
  volume = { 79 },
  year = { 2007 },
  journal = { Anal. Chem. },
  title = { {R}eal-time rolling circle amplification for protein detection },
  author = { Yang and Fung and Cho and Ellington },
}

@article{pmid17443876,
  abstract = { In vitro selection has proven to be a useful means of explore the molecules and catalysts that may have existed in a primordial 'RNA world'. By selecting binding species (aptamers) and catalysts (ribozymes) from random sequence pools, the relationship between biopolymer complexity and function can be better understood, and potential evolutionary transitions between functional molecules can be charted. In this review, we have focused on several critical events or transitions in the putative RNA world: RNA self-replication; the synthesis and utilization of nucleotide-based cofactors; acyl-transfer reactions leading to peptide and protein synthesis; and the basic metabolic pathways that are found in modern living systems. },
  month = { Apr },
  pages = { 633--655 },
  number = { 4 },
  volume = { 4 },
  year = { 2007 },
  journal = { Chem. Biodivers. },
  title = { {R}ibozyme catalysis of metabolism in the {R}{N}{A} world },
  author = { Chen and Li and Ellington },
}

@article{pmid17458167,
  abstract = { While nucleic acid binding species (aptamers) have previously been selected against a variety of biomedically important protein targets, the selection of aptamers against complex targets, such as cell surfaces and organisms, is becoming increasingly feasible. A number of aptamers have now been generated that can target specific cell-surface antigens and cell types, including tumor biomarkers and tumor cells. The generation of anti-cell aptamers has important implications for identifying disease-specific biomarkers, generating diagnostics, and developing novel drug delivery strategies. },
  month = { Apr },
  pages = { 137--144 },
  number = { 2 },
  volume = { 9 },
  year = { 2007 },
  journal = { Curr. Opin. Mol. Ther. },
  title = { {U}sing aptamers to identify and enter cells },
  author = { Chu and Ebright and Ellington },
}

@article{pmid17649966,
  month = { Jul },
  pages = { 445--448 },
  number = { 7 },
  volume = { 2 },
  year = { 2007 },
  journal = { ACS Chem. Biol. },
  title = { {W}hat's so great about {R}{N}{A}? },
  author = { Ellington },
}

@article{pmid16495043,
  abstract = { Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging. },
  month = { Apr },
  pages = { 1859--1866 },
  number = { 10 },
  volume = { 21 },
  year = { 2006 },
  journal = { Biosens Bioelectron },
  title = { {L}abeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates },
  author = { Chu and Shieh and Lavery and Levy and Richards-Kortum and Korgel and Ellington },
}

@article{pmid16507139,
  abstract = { We have developed spotted cell microarrays for measuring cellular phenotypes on a large scale. Collections of cells are printed, stained for subcellular features, then imaged via automated, high-throughput microscopy, allowing systematic phenotypic characterization. We used this technology to identify genes involved in the response of yeast to mating pheromone. Besides morphology assays, cell microarrays should be valuable for high-throughput in situ hybridization and immunoassays, enabling new classes of genetic assays based on cell imaging. },
  pages = { R6 },
  number = { 1 },
  volume = { 7 },
  year = { 2006 },
  journal = { Genome Biol. },
  title = { {S}ystematic profiling of cellular phenotypes with spotted cell microarrays reveals mating-pheromone response genes },
  author = { Narayanaswamy and Niu and Scouras and Hart and Davies and Ellington and Iyer and Marcotte },
}

@article{pmid16594629,
  abstract = { Nucleic acids can modulate gene function by base-pairing, via the molecular recognition of proteins and metabolites, and by catalysis. This diversity of functions can be combined with the ability to engineer nucleic acids based on Watson-Crick base-pairing rules to create a modular set of molecular "tools" for biotechnological and medical interventions in cellular metabolism. However, these individual RNA-based tools are most powerful when combined into rational logical or regulatory circuits, and the circuits can in turn be evolved for optimal function. Examples of genetic circuits that control translation and transcription are herein detailed, and more complex circuits with medical applications are anticipated. },
  pages = { 423--445 },
  number = { 173 },
  year = { 2006 },
  journal = { Handb Exp Pharmacol },
  title = { {E}ngineering {R}{N}{A}-based circuits },
  author = { Narayanaswamy and Ellington },
}

@article{pmid16607663,
  month = { May },
  pages = { 3338--3341 },
  number = { 20 },
  volume = { 45 },
  year = { 2006 },
  journal = { Angew. Chem. Int. Ed. Engl. },
  title = { {F}eature multiplexing--improving the efficiency of microarray devices },
  author = { Schmid and Manthiram and Grayson and Willson and Meiring and Bell and Ellington and Willson },
}

@article{pmid16621675,
  abstract = { Aptamers are selected nucleic acid binding species with affinities and specificities for protein targets that rival those of monoclonal antibodies. Furthermore, aptamers have definite advantages over antibodies, in that they can be chemically synthesized and modifications can be introduced that improve their stabilities and pharmacokinetic properties. A number of aptamers against therapeutically important targets have shown efficacy in cell and animal models, and a handful of aptamers are now in clinical trials or are being used as drugs. Recent advances in selection technologies and a more thorough exploration of how to deliver nucleic acids to target cells and tissues should further speed the process of drug development. },
  month = { Jun },
  pages = { 282--289 },
  number = { 3 },
  volume = { 10 },
  year = { 2006 },
  journal = { Curr Opin Chem Biol },
  title = { {A}ptamer therapeutics advance },
  author = { Lee and Stovall and Ellington },
}

@article{pmid16648360,
  abstract = { Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can 'read' one sequence and then 'write' (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs. },
  pages = { 2166--2172 },
  number = { 8 },
  volume = { 34 },
  year = { 2006 },
  journal = { Nucleic Acids Res. },
  title = { {D}eoxyribozymes that recode sequence information },
  author = { Tabor and Levy and Ellington },
}

@article{pmid16740739,
  abstract = { Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology. },
  pages = { e73 },
  number = { 10 },
  volume = { 34 },
  year = { 2006 },
  journal = { Nucleic Acids Res. },
  title = { {A}ptamer mediated si{R}{N}{A} delivery },
  author = { Chu and Twu and Ellington and Levy },
}

@article{pmid16778167,
  abstract = { We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Aptamer:toxin conjugates have an IC50 of 27 nmol/L and display an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer not only promotes uptake into target cells but also decreases the toxicity of gelonin in non-target cells. These results validate the notion that "escort aptamers" may be useful for the treatment of specific tumors expressing unique antigen targets. },
  month = { Jun },
  pages = { 5989--5992 },
  number = { 12 },
  volume = { 66 },
  year = { 2006 },
  journal = { Cancer Res. },
  title = { {A}ptamer:toxin conjugates that specifically target prostate tumor cells },
  author = { Chu and Marks and Lavery and Faulkner and Rosenblum and Ellington and Levy },
}

@article{pmid16834439,
  abstract = { DNAzymes are catalytically active DNA molecules, which have previously been described in solution. Here, we organize these molecules into a series of two-dimensional (2D) arrays using a periodic arrangement of DNA structures based on the DNA double crossover motif. We demonstrate by means of atomic force microscopy that the DNAzymes are organized according to the design and that they retain their activity when attached in linear strings within the context of the 2D array. },
  month = { Jul },
  pages = { 1505--1507 },
  number = { 7 },
  volume = { 6 },
  year = { 2006 },
  journal = { Nano Lett. },
  title = { {F}unctional {D}{N}{A}zymes organized into two-dimensional arrays },
  author = { Garibotti and Knudsen and Ellington and Seeman },
}

@article{pmid17165697,
  abstract = { Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes. },
  month = { Dec },
  pages = { 15936--15937 },
  number = { 50 },
  volume = { 128 },
  year = { 2006 },
  journal = { J. Am. Chem. Soc. },
  title = { {R}ibozyme-mediated signal augmentation on a mass-sensitive biosensor },
  author = { Knudsen and Lee and Ellington and Savran },
}

@article{pmid17723365,
  abstract = { Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics. },
  month = { Mar },
  pages = { 82--90 },
  number = { 1 },
  volume = { 564 },
  year = { 2006 },
  journal = { Anal. Chim. Acta },
  title = { {O}ptimization of aptamer microarray technology for multiple protein targets },
  author = { Cho and Collett and Szafranska and Ellington },
}

@article{pmid18476324,
  pages = { 21--44 },
  volume = { 22 },
  year = { 2006 },
  journal = { Biotechnol. Genet. Eng. Rev. },
  title = { {D}eveloping {R}{N}{A} tools for engineered regulatory systems },
  author = { Tabor and Davidson and Ellington },
}

@article{pmid15677335,
  month = { Feb },
  pages = { 1273--1274 },
  number = { 5 },
  volume = { 102 },
  year = { 2005 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {M}istakes in translation don't translate into termination },
  author = { Hughes and Ellington },
}

@article{pmid15707941,
  abstract = { High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips. },
  month = { Mar },
  pages = { 113--123 },
  number = { 1 },
  volume = { 338 },
  year = { 2005 },
  journal = { Anal. Biochem. },
  title = { {F}unctional {R}{N}{A} microarrays for high-throughput screening of antiprotein aptamers },
  author = { Collett and Cho and Lee and Levy and Hood and Wan and Ellington },
}

@article{pmid15713061,
  abstract = { An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 muM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified "switch" that was responsive to ATP concentration. },
  month = { Feb },
  pages = { 2022--2023 },
  number = { 7 },
  volume = { 127 },
  year = { 2005 },
  journal = { J. Am. Chem. Soc. },
  title = { U}sing a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, {A}{T}{P },
  author = { Cho and Yang and Levy and Ellington },
}

@article{pmid15734551,
  abstract = { RNA has long been a favoured medium for in vitro evolution and engineering. Functional RNAs produced in vitro can bind small molecules (aptamers), possess catalytic activity (ribozymes) or do both (aptazymes). A plethora of recent work has shown similar strategies used naturally for gene regulation in bacteria. Interest in these natural systems has inspired an effort to engineer and evolve this activity in vivo. A recent paper by Isaacs et al. describes the engineering and in vivo activity of a small RNA that removes translation inhibition by binding the 5' untranslated region of its target mRNA and making the ribosome-binding site accessible. },
  month = { Mar },
  pages = { 109--112 },
  number = { 3 },
  volume = { 23 },
  year = { 2005 },
  journal = { Trends Biotechnol. },
  title = { {E}ngineering regulatory {R}{N}{A}s },
  author = { Davidson and Ellington },
}

@article{pmid15769669,
  abstract = { Most biopolymer drugs to date have been proteins. However, the ability to select nucleic acid binding species (aptamers) has led to the development of protein inhibitors and modulators that are small, readily synthesized nucleic acids. The techniques for optimizing, stabilizing, and delivering nucleic acid therapies are just beginning to be developed, but the same engineering flexibility that has so far allowed the generation of multiple, high affinity and specificity binding species appears to also apply to the methods for adapting nucleic acids to clinical applications. We review the selection and characterization of various aptamers and their applications to a variety of disease states, and then focus on the hurdles that must be overcome for the use of aptamers as both exogenously delivered drugs and as gene therapies. },
  pages = { 1802--1827 },
  volume = { 10 },
  year = { 2005 },
  journal = { Front. Biosci. },
  title = { {A}ptamers: prospects in therapeutics and biomedicine },
  author = { Yan and Bell and Breeden and Ellington },
}

@article{pmid16012175,
  abstract = { Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+. },
  month = { Aug },
  pages = { 369--377 },
  number = { 8 },
  volume = { 18 },
  year = { 2005 },
  journal = { Protein Eng. Des. Sel. },
  title = { {A} modified consensus approach to mutagenesis inverts the cofactor specificity of {B}acillus stearothermophilus lactate dehydrogenase },
  author = { Flores and Ellington },
}

@article{pmid16131588,
  abstract = { We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically may have been the only active variant in the starting pool. The isolation of this sequence from the population suggests that this selection method is extremely robust at selecting optimal ribozymes and should, therefore, prove useful for the selection and optimization of other trans-acting nucleic acid catalysts capable of multiple turnover catalysis. },
  month = { Oct },
  pages = { 1555--1562 },
  number = { 10 },
  volume = { 11 },
  year = { 2005 },
  journal = { RNA },
  title = { {D}irect selection of trans-acting ligase ribozymes by in vitro compartmentalization },
  author = { Levy and Griswold and Ellington },
}

@article{pmid16199170,
  abstract = { Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5' RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer microarrays, including blocking, washing, drying, and scanning. Examples are shown for the specific capture of fluorescently labeled target proteins either alone in binding buffer or in competition with labeled intracellular proteins from cell lysates. Consideration is given to the challenges involved in producing multiplex aptamer chips composed of aptamers taken from disparate literature sources, and to the development of standardized methods for characterizing the performance of capture reagents used in biosensors. },
  month = { Sep },
  pages = { 4--15 },
  number = { 1 },
  volume = { 37 },
  year = { 2005 },
  journal = { Methods },
  title = { {P}roduction and processing of aptamer microarrays },
  author = { Collett and Cho and Ellington },
}

@article{pmid16217837,
  abstract = { Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties. },
  month = { Oct },
  pages = { 1327--1337 },
  number = { 10 },
  volume = { 40 },
  year = { 2005 },
  journal = { J Mass Spectrom },
  title = { {E}lectrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity },
  author = { Keller and Breeden and Zhang and Ellington and Brodbelt },
}

@article{pmid16237122,
  abstract = { The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins. },
  pages = { e162 },
  number = { 18 },
  volume = { 33 },
  year = { 2005 },
  journal = { Nucleic Acids Res. },
  title = { {P}roximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores },
  author = { Pai and Ellington and Levy },
}

@article{pmid16239467,
  month = { Oct },
  pages = { 454--455 },
  number = { 5747 },
  volume = { 310 },
  year = { 2005 },
  journal = { Science },
  title = { {E}volution. {C}hanging the cofactor diet of an enzyme },
  author = { Ellington and Bull },
}

@article{pmid16246910,
  abstract = { Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding. },
  pages = { 6090--6100 },
  number = { 19 },
  volume = { 33 },
  year = { 2005 },
  journal = { Nucleic Acids Res. },
  title = { {B}inding of herpes simplex virus-1 {U}{S}11 to specific {R}{N}{A} sequences },
  author = { Bryant and Cox and Wang and Hogle and Ellington and Coen },
}

@article{pmid16254932,
  month = { Dec },
  pages = { 2163--2166 },
  number = { 12 },
  volume = { 6 },
  year = { 2005 },
  journal = { Chembiochem },
  title = { {Q}uantum-dot aptamer beacons for the detection of proteins },
  author = { Levy and Cater and Ellington },
}

@article{pmid16306980,
  abstract = { We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps. },
  month = { Nov },
  pages = { 441--442 },
  number = { 7067 },
  volume = { 438 },
  year = { 2005 },
  journal = { Nature },
  title = { {S}ynthetic biology: engineering {E}scherichia coli to see light },
  author = { Levskaya and Chevalier and Tabor and Simpson and Lavery and Levy and Davidson and Scouras and Ellington and Marcotte and Voigt },
}

@article{pmid16314457,
  abstract = { A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins. },
  month = { Dec },
  pages = { 1848--1857 },
  number = { 12 },
  volume = { 11 },
  year = { 2005 },
  journal = { RNA },
  title = { A}rginine-rich motifs present multiple interfaces for specific binding by {R}{N}{A },
  author = { Bayer and Booth and Knudsen and Ellington },
}

@article{pmid14681367,
  abstract = { The aptamer database is designed to contain comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods. Such data are not normally collected in 'natural' sequence databases, such as GenBank. Besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility, the database serves as a valuable resource for theoretical biologists who describe and explore fitness landscapes. The database is updated monthly and is publicly available at http://aptamer. icmb.utexas.edu/. },
  month = { Jan },
  pages = { 95--100 },
  number = { Database issue },
  volume = { 32 },
  year = { 2004 },
  journal = { Nucleic Acids Res. },
  title = { {A}ptamer database },
  author = { Lee and Hesselberth and Meyers and Ellington },
}

@article{pmid14681388,
  abstract = { We have created an Amino Acid-Nucleotide Interaction Database (AANT; http://aant.icmb.utexas. edu/) that categorizes all amino acid-nucleotide interactions from experimentally determined protein-nucleic acid structures, and provides users with a graphic interface for visualizing these interactions in aggregate. AANT accomplishes this by extracting individual amino acid-nucleotide interactions from structures in the Protein Data Bank, combining and superimposing these interactions into multiple structure files (e.g. 20 amino acids x 5 nucleotides) and grouping structurally similar interactions into more readily identifiable clusters. Using the Chime web browser plug-in, users can view 3D representations of the superimpositions and clusters. The unique collection and representation of data on amino acid-nucleotide interactions facilitates understanding the specificity of protein-nucleic acid interactions at a more fundamental level, and allows comparison of otherwise extremely disparate sets of structures. Moreover, by modularly representing the fundamental interactions that govern binding specificity it may prove possible to better engineer nucleic acid binding proteins. },
  month = { Jan },
  pages = { D174--181 },
  number = { Database issue },
  volume = { 32 },
  year = { 2004 },
  journal = { Nucleic Acids Res. },
  title = { {A}{A}{N}{T}: the {A}mino {A}cid-{N}ucleotide {I}nteraction {D}atabase },
  author = { Hoffman and Khrapov and Cox and Yao and Tong and Ellington },
}

@article{pmid14681590,
  abstract = { A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays). },
  month = { Jan },
  pages = { 114--127 },
  number = { 1 },
  volume = { 10 },
  year = { 2004 },
  journal = { RNA },
  title = { {I}n vitro selection of ribozymes dependent on peptides for activity },
  author = { Robertson and Knudsen and Ellington },
}

@article{pmid15048102,
  month = { Apr },
  pages = { 301--303 },
  number = { 4 },
  volume = { 11 },
  year = { 2004 },
  journal = { Nat. Struct. Mol. Biol. },
  title = { {R}ibozyme déjà vu },
  author = { Knudsen and Ellington },
}

@article{pmid15167801,
  abstract = { We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution. },
  month = { Jun },
  pages = { 3194--3198 },
  number = { 11 },
  volume = { 76 },
  year = { 2004 },
  journal = { Anal. Chem. },
  title = { {M}icromechanical detection of proteins using aptamer-based receptor molecules },
  author = { Savran and Knudsen and Ellington and Manalis },
}

@article{pmid15253644,
  abstract = { Aptamer biosensors have been immobilized on beads, introduced into micromachined chips on the electronic tongue sensor array, and used for the detection and quantitation of proteins. Aptamer chips could detect proteins in both capture and sandwich assay formats. Unlike most protein-based arrays, the aptamer chips could be stripped and reused multiple times. The aptamer chips proved to be useful for screening aptamers from in vitro selection experiments and for sensitively quantitating the biothreat agent ricin. },
  month = { Jul },
  pages = { 4066--4075 },
  number = { 14 },
  volume = { 76 },
  year = { 2004 },
  journal = { Anal. Chem. },
  title = { {A}ptamer-based sensor arrays for the detection and quantitation of proteins },
  author = { Kirby and Cho and Gehrke and Bayer and Park and Neikirk and McDevitt and Ellington },
}

@article{pmid15300257,
  abstract = { Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior. In particular, modifications at the 2'-OH of ribose are important for enhancing the stability of RNA. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2' position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript. },
  month = { Sep },
  pages = { 1155--1160 },
  number = { 9 },
  volume = { 22 },
  year = { 2004 },
  journal = { Nat. Biotechnol. },
  title = { E}volution of a {T}7 {R}{N}{A} polymerase variant that transcribes 2'-{O}-methyl {R}{N}{A },
  author = { Chelliserrykattil and Ellington },
}

@article{pmid15461425,
  abstract = { Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process. },
  month = { Jun },
  pages = { 681--691 },
  number = { 6 },
  volume = { 58 },
  year = { 2004 },
  journal = { J. Mol. Evol. },
  title = { {T}he robustness of naturally and artificially selected nucleic acid secondary structures },
  author = { Meyers and Lee and Cowperthwaite and Ellington },
}

@article{pmid15556407,
  abstract = { In vitro selection experiments have clearly demonstrated that RNA can perform many of the functions necessary to support an RNA world. Moreover, it appears that novel functions could have readily evolved from existing functional RNA molecules. Therefore, diverse molecular ecosystems could potentially have arisen from an initial, small population of functional replicators. These findings suggest that the sequences of living systems may have been determined in part by chance occurrences at origins. Any extrapolations linking sequences (as opposed to functions) obtained in the laboratory to what may have occurred ca. 4 billion years ago are tenuous at best. Thus, perhaps the best way to understand origins is not by examining relatively unconstrained sequence information, but by examining the inherent constraints imposed by prebiotic chemistry. },
  month = { Dec },
  pages = { 629--633 },
  number = { 6 },
  volume = { 8 },
  year = { 2004 },
  journal = { Curr Opin Chem Biol },
  title = { {T}he importance of prebiotic chemistry in the {R}{N}{A} world },
  author = { Hughes and Robertson and Ellington and Levy },
}

@article{pmid15600355,
  abstract = { Because of their relative simplicity, synthetic receptors often lack the selectivity observed for biopolymer receptors, such as aptamers. However, aptamer recognition of ligands is limited by the chemistries inherent in the four canonical nucleotides. Here, we report the design and selection of a ternary complex in which the specificity of a bis-boronic acid synthetic host (1) that binds to various carboxylic acids is tuned by a surrounding aptamer. Although, the synthetic receptor alone has higher selectivity for citrate over DL-tartrate, the formation of the aptamer:receptor complex reversed the organic host selectivity to preferentially bind tartrate. The RNA conformation changed upon the introduction of the synthetic host, consistent with an induced-fit mechanism for binding. },
  month = { Dec },
  pages = { 16515--16519 },
  number = { 50 },
  volume = { 126 },
  year = { 2004 },
  journal = { J. Am. Chem. Soc. },
  title = { {T}uning the specificity of a synthetic receptor using a selected nucleic acid receptor },
  author = { Manimala and Wiskur and Ellington and Anslyn },
}

@article{pmid16701231,
  abstract = { Although the genetic code is almost universal, natural variations exist that have caused evolutionary biologists to speculate about codon evolution. There are two predominant hypotheses that specify either a gradual (ambiguous intermediate) or stochastic (codon capture) change in the code. These hypotheses are similar to two biotechnology techniques that have been used to engineer the genetic code: a 'top down' approach, in which the whole organism is evolved for the ability to incorporate unnatural amino acids, and a 'bottom up' approach, in which aminoacyl-tRNA synthetases and their cognate tRNAs are engineered. The biotechnology experiments provide insights into natural codon evolution, and a combination of these approaches should enable the evolution of organisms that can incorporate unnatural amino acids throughout their proteomes. },
  month = { Feb },
  pages = { 69--75 },
  number = { 2 },
  volume = { 19 },
  year = { 2004 },
  journal = { Trends Ecol. Evol. (Amst.) },
  title = { {E}volving new genetic codes },
  author = { Bacher and Hughes and Tze-Fei Wong and Ellington },
}

@article{pmid12531194,
  abstract = { Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection. },
  month = { Jan },
  pages = { 106--112 },
  number = { 2 },
  volume = { 312 },
  year = { 2003 },
  journal = { Anal. Biochem. },
  title = { {S}imultaneous detection of diverse analytes with an aptazyme ligase array },
  author = { Hesselberth and Robertson and Knudsen and Ellington },
}

@article{pmid12698297,
  abstract = { Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated with a 5'-iodine and a 3'phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that peptide and nucleic acid replicators could have been interdependent. },
  month = { May },
  pages = { 607--615 },
  number = { 5 },
  volume = { 56 },
  year = { 2003 },
  journal = { J. Mol. Evol. },
  title = { {P}eptide-templated nucleic acid ligation },
  author = { Levy and Ellington },
}

@article{pmid12743371,
  abstract = { We have designed an autocatalytic cycle based on the highly efficient 10-23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10-23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an in vitro selection experiment conducted in the absence of any protein enzymes. },
  month = { May },
  pages = { 6416--6421 },
  number = { 11 },
  volume = { 100 },
  year = { 2003 },
  journal = { Proc. Natl. Acad. Sci. U.S.A. },
  title = { {E}xponential growth by cross-catalytic cleavage of deoxyribozymogens },
  author = { Levy and Ellington },
}

@article{pmid12824567,
  pages = { 27--43 },
  volume = { 230 },
  year = { 2003 },
  journal = { Methods Mol. Biol. },
  title = { {A}utogene selections },
  author = { Chelliserrykattil and Ellington },
}

@article{pmid12949563,
  month = { Sep },
  pages = { 1013--1015 },
  number = { 9 },
  volume = { 21 },
  year = { 2003 },
  journal = { Nat. Biotechnol. },
  title = { {P}laying to win at {D}{N}{A} computation },
  author = { Tabor and Ellington },
}

@article{pmid14500834,
  abstract = { While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes. },
  month = { Oct },
  pages = { 5700--5713 },
  number = { 19 },
  volume = { 31 },
  year = { 2003 },
  journal = { Nucleic Acids Res. },
  title = { {I}n vitro selection of molecular beacons },
  author = { Rajendran and Ellington },
}

@article{pmid14667253,
  abstract = { The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.\\ The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.\\ A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates. },
  month = { Dec },
  pages = { 24 },
  volume = { 3 },
  year = { 2003 },
  journal = { BMC Evol. Biol. },
  title = { {E}volution of phage with chemically ambiguous proteomes },
  author = { Bacher and Bull and Ellington },
}

@article{pmid14674448,
  abstract = { The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system. },
  month = { Sep },
  pages = { 4732--4739 },
  number = { 18 },
  volume = { 75 },
  year = { 2003 },
  journal = { Anal. Chem. },
  title = { {D}{N}{A} hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays },
  author = { Ali and Kirby and Goodey and Rodriguez and Ellington and Neikirk and McDevitt },
}

@article{pmid11768971,
  pages = { 259--267 },
  volume = { 182 },
  year = { 2002 },
  journal = { Methods Mol. Biol. },
  title = { {M}utagenic polymerase chain reaction of protein-coding genes for in vitro evolution },
  author = { Matsumura and Ellington },
}

@article{pmid11786015,
  abstract = { The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures. },
  month = { Jan },
  pages = { 325--337 },
  number = { 3 },
  volume = { 315 },
  year = { 2002 },
  journal = { J. Mol. Biol. },
  title = { {I}ncreasing the thermal stability of an oligomeric protein, beta-glucuronidase },
  author = { Flores and Ellington },
}

@article{pmid11821911,
  abstract = { Deoxyribozymes that could catalyze the formation of an internucleotide phosphorothioester linkage were selected from a random sequence pool. During the course of the selection, the pool was successively challenged with five oligonucleotide substrates, each of which terminated in the same hexanucleotide sequence. Selected deoxyribozyme ligases could use all five substrates, albeit to different degrees, and appeared to form secondary structures that allow differential pairing between the deoxyribozyme and each substrate. These results suggest that early replicases may have been able to bind a variety of oligonucleotide substrates while catalyzing ligation via a common junction. },
  month = { Feb },
  pages = { 180--190 },
  number = { 2 },
  volume = { 54 },
  year = { 2002 },
  journal = { J. Mol. Evol. },
  title = { {I}n vitro selection of a deoxyribozyme that can utilize multiple substrates },
  author = { Levy and Ellington },
}

@article{pmid11983331,
  abstract = { Effector-activated ribozymes that respond to small organic molecules have previously been generated by appending binding species (aptamers) to ribozymes. In order to determine if deoxyribozymes can similarly be activated by effector molecules, we have appended an anti-adenosine aptamer to a selected deoxyribozyme ligase. The resultant constructs are specifically activated by ATP. Optimization of the joining region resulted in ligases that are activated up to 460-fold by ATP. The selected deoxyribozyme catalyzes ligation largely via a templating mechanism. Effector activation is surprisingly achieved by suppression of the rate of the background, templated ligation reaction in the absence of the effector molecule, probably by misalignment of the oligonucleotide substrates. This novel allosteric mechanism has not previously been observed for nucleic-acid catalysts and is rare even in protein catalysts. },
  month = { Apr },
  pages = { 417--426 },
  number = { 4 },
  volume = { 9 },
  year = { 2002 },
  journal = { Chem. Biol. },
  title = { {A}{T}{P}-dependent allosteric {D}{N}{A} enzymes },
  author = { Levy and Ellington },
}

@article{pmid12052178,
  abstract = { In vitro selection can be used to generate nucleic acid binding species (aptamers) and catalysts (ribozymes) that can recognize a variety of molecules. Because nucleic acid function is largely derived from readily tabulated secondary structures, it has proven possible to engineer aptamers and ribozymes to function as biosensors. Labeling nucleic acids with reporter molecules has yielded simple antibody substitutes, but by relying on ligand-dependent conformational changes it has also proven possible to generate biosensors that can recognize and specifically report the presence of ligands in homogenous solution. It may prove possible to generate signaling aptamers and allosteric ribozymes (aptazymes) that are responsive to a large fraction of an organismal proteome or metabolome using automated methods. Nucleic acid biosensor arrays for non-nucleic acid targets could likely be generated with the same facility as DNA chips. },
  month = { Jun },
  pages = { 263--270 },
  number = { 4 },
  volume = { 5 },
  year = { 2002 },
  journal = { Comb. Chem. High Throughput Screen. },
  title = { {S}electing nucleic acids for biosensor applications },
  author = { Rajendran and Ellington },
}

@article{pmid12052180,
  abstract = { While the in vitro selection of nucleic acid binding species (aptamers) requires numerous liquid-handling steps, these steps are relatively straightforward and the overall process is therefore amenable to automation. Here we demonstrate that automated selection techniques are capable of generating aptamers against a number of diverse protein targets. Automated selection techniques can be integrated with automated analytical methods, including sequencing, determination of binding constants, and structural analysis. The methods that have so far been developed can be further multiplexed, and it should soon be possible to attempt the selection of aptamers against organismal proteomes or metabolomes. },
  month = { Jun },
  pages = { 289--299 },
  number = { 4 },
  volume = { 5 },
  year = { 2002 },
  journal = { Comb. Chem. High Throughput Screen. },
  title = { {A}utomated acquisition of aptamer sequences },
  author = { Cox and Rajendran and Riedel and Davidson and Sooter and Bayer and Schmitz-Brown and Ellington },
}

@article{pmid12089558,
  abstract = { Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process. },
  month = { Jul },
  pages = { 717--722 },
  number = { 7 },
  volume = { 20 },
  year = { 2002 },
  journal = { Nat. Biotechnol. },
  title = { {P}rotein-dependent ribozymes report molecular interactions in real time },
  author = { Hartig and Najafi-Shoushtari and Grune and Yan and Ellington and Famulok },
}

@article{pmid12165173,
  pages = { 339--346 },
  number = { 3 },
  volume = { 4 },
  year = { 2002 },
  journal = { Diabetes Technol. Ther. },
  title = { {A}ptamers as potential diagnostic reagents for diabetes },
  author = { Yan and Ellington },
}

@article{pmid12186650,
  abstract = { DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions. },
  month = { Jul },
  pages = { REVIEWS1021 },
  number = { 8 },
  volume = { 3 },
  year = { 2002 },
  journal = { Genome Biol. },
  title = { {A}nticipatory evolution and {D}{N}{A} shuffling },
  author = { Bacher and Reiss and Ellington },
}

@article{pmid12204684,
  abstract = { Aptamers composed of L-nucleic acids, Spiegelmers, were selected to specifically bind GnRH. Spiegelmer inhibition of GnRH activity was demonstrated in both cellular and animal models. Rabbit studies showed minimal immunogenic response to the agents. },
  month = { Aug },
  pages = { 857--858 },
  number = { 8 },
  volume = { 9 },
  year = { 2002 },
  journal = { Chem. Biol. },
  title = { {R}eflections on a novel therapeutic candidate },
  author = { Sooter and Ellington },
}

@article{pmid12384610,
  abstract = { Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands. },
  month = { Oct },
  pages = { e108 },
  number = { 20 },
  volume = { 30 },
  year = { 2002 },
  journal = { Nucleic Acids Res. },
  title = { {A}utomated selection of aptamers against protein targets translated in vitro: from gene to aptamer },
  author = { Cox and Hayhurst and Hesselberth and Bayer and Georgiou and Ellington },
}

@article{pmid12447352,
  month = { Dec },
  pages = { 891--893 },
  number = { 12 },
  volume = { 9 },
  year = { 2002 },
  journal = { Nat. Struct. Biol. },
  title = { {A} (ribo) switch in the paradigms of genetic regulation },
  author = { Hesselberth and Ellington },
}

@article{pmid12466025,
  abstract = { Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo.\\ By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules.\\ Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies. },
  month = { Dec },
  pages = { 21 },
  volume = { 2 },
  year = { 2002 },
  journal = { BMC Biotechnol. },
  title = { {G}roup {I} aptazymes as genetic regulatory switches },
  author = { Thompson and Syrett and Knudsen and Ellington },
}

@article{pmid12466564,
  abstract = { Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins. },
  month = { Dec },
  pages = { e132 },
  number = { 23 },
  volume = { 30 },
  year = { 2002 },
  journal = { Nucleic Acids Res. },
  title = { {C}onstruction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid {P}{C}{R}-based sequencing },
  author = { Yang and Bassett and Li and Luxon and Herzog and Shope and Aronson and Prow and Leary and Kirby and Ellington and Gorenstein },
}

@article{pmid18428900,
  abstract = { The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool. },
  month = { Feb },
  pages = { Unit 9.6 },
  volume = { Chapter 9 },
  year = { 2002 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {I}n vitro selection using modified or unnatural nucleotides },
  author = { Knudsen and Robertson and Ellington },
}

@article{pmid11124909,
  abstract = { The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity. },
  month = { Jan },
  pages = { 331--339 },
  number = { 2 },
  volume = { 305 },
  year = { 2001 },
  journal = { J. Mol. Biol. },
  title = { {I}n vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates },
  author = { Matsumura and Ellington },
}

@article{pmid11252780,
  month = { Mar },
  pages = { 474--476 },
  number = { 3 },
  volume = { 30 },
  year = { 2001 },
  journal = { BioTechniques },
  title = { {O}ptimization of heterologous gene expression for in vitro evolution },
  author = { Matsumura and Olsen and Ellington },
}

@article{pmid11283579,
  month = { Apr },
  pages = { 313--314 },
  number = { 4 },
  volume = { 19 },
  year = { 2001 },
  journal = { Nat. Biotechnol. },
  title = { {S}witching nucleic acids for antibodies },
  author = { Hoffman and Hesselberth and Ellington },
}

@article{pmid11345430,
  abstract = { A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results. },
  month = { Apr },
  pages = { 513--523 },
  number = { 4 },
  volume = { 7 },
  year = { 2001 },
  journal = { RNA },
  title = { {O}ptimization and optimality of a short ribozyme ligase that joins non-{W}atson-{C}rick base pairings },
  author = { Robertson and Hesselberth and Ellington },
}

@article{pmid11369241,
  month = { May },
  pages = { R336 },
  number = { 9 },
  volume = { 11 },
  year = { 2001 },
  journal = { Curr. Biol. },
  title = { {D}{N}{A} computation function },
  author = { Cox and Ellington },
}

@article{pmid11401165,
  abstract = { To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base. },
  month = { Jun },
  pages = { 744--753 },
  number = { 6 },
  volume = { 12 },
  year = { 2001 },
  journal = { J. Am. Soc. Mass Spectrom. },
  title = { {E}lucidation of the initial step of oligonucleotide fragmentation in matrix-assisted laser desorption/ionization using modified nucleic acids },
  author = { Christian and Reilly and Mokler and Wincott and Ellington },
}

@article{pmid11427883,
  month = { Jul },
  pages = { 580--582 },
  number = { 7 },
  volume = { 8 },
  year = { 2001 },
  journal = { Nat. Struct. Biol. },
  title = { {T}he descent of polymerization },
  author = { Levy and Ellington },
}

@article{pmid11433277,
  abstract = { Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome. },
  month = { Jul },
  pages = { 650--655 },
  number = { 7 },
  volume = { 19 },
  year = { 2001 },
  journal = { Nat. Biotechnol. },
  title = { {I}n vitro selection of nucleoprotein enzymes },
  author = { Robertson and Ellington },
}

@article{pmid11514527,
  abstract = { Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues. },
  month = { Sep },
  pages = { 5414--5425 },
  number = { 18 },
  volume = { 183 },
  year = { 2001 },
  journal = { J. Bacteriol. },
  title = { {S}election and characterization of {E}scherichia coli variants capable of growth on an otherwise toxic tryptophan analogue },
  author = { Bacher and Ellington },
}

@article{pmid11525762,
  abstract = { The recent selection of a complex ribozyme capable of general polymerization on a template in trans has revealed how catalysts may have arisen from one another in the RNA world. },
  month = { Aug },
  pages = { R665--667 },
  number = { 16 },
  volume = { 11 },
  year = { 2001 },
  journal = { Curr. Biol. },
  title = { {R}{N}{A} world: catalysis abets binding, but not vice versa },
  author = { Levy and Ellington },
}

@article{pmid11557339,
  abstract = { The in vitro selection of nucleic acid binding species (aptamers) is frequently repetitive, time-consuming, and poorly adapted to high-throughput applications. We have adapted automated workstations to select anti-protein aptamers; as an example, we demonstrated the selection of anti-lysozyme aptamers that function as efficient inhibitors of cell lysis. The increases in throughput brought about by automation should potentiate the application of aptamer technology to the rapidly growing field of proteomics. },
  month = { Oct },
  pages = { 2525--2531 },
  number = { 10 },
  volume = { 9 },
  year = { 2001 },
  journal = { Bioorg. Med. Chem. },
  title = { {A}utomated selection of anti-protein aptamers },
  author = { Cox and Ellington },
}

@article{pmid11557346,
  abstract = { The chemical ligation of DNA molecules can be mediated by terminal phosphorothioate displacement of a 5' iodine. We have selected deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages. The selected deoxyribozymes enhance the rate of ligation in part through the provision of a template that aligns the ligation junction and do not appear to require metal ions for catalysis. },
  month = { Oct },
  pages = { 2581--2587 },
  number = { 10 },
  volume = { 9 },
  year = { 2001 },
  journal = { Bioorg. Med. Chem. },
  title = { {S}election of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage },
  author = { Levy and Ellington },
}

@article{pmid11688716,
  abstract = { Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress. },
  month = { Sep },
  pages = { 1327--1334 },
  number = { 9 },
  volume = { 382 },
  year = { 2001 },
  journal = { Biol. Chem. },
  title = { {T}oward automated nucleic acid enzyme selection },
  author = { Sooter and Riedel and Davidson and Levy and Cox and Ellington },
}

@article{pmid11806761,
  abstract = { The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.\\ A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides. },
  pages = { 13 },
  volume = { 1 },
  year = { 2001 },
  journal = { BMC Biotechnol. },
  title = { {A} combined in vitro/in vivo selection for polymerases with novel promoter specificities },
  author = { Chelliserrykattil and Cai and Ellington },
}

@article{pmid18265209,
  abstract = { This unit describes the design, synthesis, and amplification of random-sequence DNA pools, from which functional nucleic acid-binding or catalytic species can be selected. Since it is an expensive and time-consuming process, the authors have provided an extensive strategic planning section to guide investigators in designing and constructing the pool. },
  month = { May },
  pages = { Unit 24.2 },
  volume = { Chapter 24 },
  year = { 2001 },
  journal = { Curr Protoc Mol Biol },
  title = { {D}esign, synthesis, and amplification of {D}{N}{A} pools for construction of combinatorial pools and libraries },
  author = { Pollard and Bell and Ellington },
}

@article{pmid18265210,
  abstract = { In vitro selection of RNA aptamers that bind to a protein target is detailed in the protocols presented in this unit. Aptamers generated from these types of selection experiments can potentially function as protein inhibitors, and are often used as diagnostic or therapeutic reagents. },
  month = { May },
  pages = { Unit 24.3 },
  volume = { Chapter 24 },
  year = { 2001 },
  journal = { Curr Protoc Mol Biol },
  title = { {I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization },
  author = { Jhaveri and Ellington },
}

@article{pmid18428880,
  abstract = { Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions. },
  month = { May },
  pages = { Unit 9.2 },
  volume = { Chapter 9 },
  year = { 2001 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection },
  author = { Pollard and Bell and Ellington },
}

@article{pmid18428881,
  abstract = { This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially function as protein inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate a ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription. },
  month = { May },
  pages = { Unit 9.3 },
  volume = { Chapter 9 },
  year = { 2001 },
  journal = { Curr Protoc Nucleic Acid Chem },
  title = { {I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization },
  author = { Jhaveri and Ellington },
}

@article{pmid10671531,
  abstract = { The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors. },
  month = { Feb },
  pages = { 4937--4942 },
  number = { 7 },
  volume = { 275 },
  year = { 2000 },
  journal = { J. Biol. Chem. },
  title = { {I}n vitro selection of {R}{N}{A} molecules that inhibit the activity of ricin {A}-chain },
  author = { Hesselberth and Miller and Robertus and Ellington },
}

@article{pmid10671532,
  abstract = { Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells. },
  month = { Feb },
  pages = { 4943--4948 },
  number = { 7 },
  volume = { 275 },
  year = { 2000 },
  journal = { J. Biol. Chem. },
  title = { {R}{N}{A} aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin },
  author = { Hirao and Madin and Endo and Yokoyama and Ellington },
}

@article{pmid10734194,
  abstract = { A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable. },
  month = { Apr },
  pages = { 1751--1759 },
  number = { 8 },
  volume = { 28 },
  year = { 2000 },
  journal = { Nucleic Acids Res. },
  title = { {D}esign and optimization of effector-activated ribozyme ligases },
  author = { Robertson and Ellington },
}

@article{pmid10786840,
  abstract = { It has been suggested that in vitro selection experiments can provide information not only on what might have occurred during the evolution of the RNA world, but can in fact yield insights into particular features of the RNA world. In particular, it has been suggested that the sequences of anti-amino acid aptamers can provide clues to the origin of the genetic code, and that there is a statistically significant association between motifs found in aptamers and codons. We argue that the suggested connections between modern motifs and ancient sequences are logically tenuous, and show that there is no statistically meaningful association between motifs found in aptamers and codons. },
  month = { Apr },
  pages = { 485--498 },
  number = { 4 },
  volume = { 6 },
  year = { 2000 },
  journal = { RNA },
  title = { {T}he scene of a frozen accident },
  author = { Ellington and Khrapov and Shaw },
}

@article{pmid10889989,
  pages = { 193--214 },
  volume = { 318 },
  year = { 2000 },
  journal = { Meth. Enzymol. },
  title = { {I}n vitro selection of {R}{N}{A} aptamers },
  author = { Marshall and Ellington },
}

@article{pmid10943569,
  abstract = { In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as 'universal receptors' that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes. },
  month = { Mar },
  pages = { 15--25 },
  number = { 1 },
  volume = { 74 },
  year = { 2000 },
  journal = { J. Biotechnol. },
  title = { {I}n vitro selection of nucleic acids for diagnostic applications },
  author = { Hesselberth and Robertson and Jhaveri and Ellington },
}

@article{pmid11101810,
  abstract = { Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals. },
  month = { Dec },
  pages = { 1293--1297 },
  number = { 12 },
  volume = { 18 },
  year = { 2000 },
  journal = { Nat. Biotechnol. },
  title = { {I}n vitro selection of signaling aptamers },
  author = { Jhaveri and Rajendran and Ellington },
}

@article{pmid10404164,
  abstract = { The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters. },
  month = { Jul },
  pages = { 696--701 },
  number = { 7 },
  volume = { 17 },
  year = { 1999 },
  journal = { Nat. Biotechnol. },
  title = { {D}irected evolution of the surface chemistry of the reporter enzyme beta-glucuronidase },
  author = { Matsumura and Wallingford and Surana and Vize and Ellington },
}

@article{pmid10433691,
  abstract = { The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure. },
  month = { Aug },
  pages = { 9850--9861 },
  number = { 31 },
  volume = { 38 },
  year = { 1999 },
  journal = { Biochemistry },
  title = { {N}{M}{R} mapping of the recombinant mouse major urinary protein {I} binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole },
  author = { Zidek and Stone and Lato and Pagel and Miao and Ellington and Novotny },
}

@article{pmid10472627,
  abstract = { The first living systems may have employed template-directed oligonucleotide ligation for replication. The utility of oligonucleotide ligation as a mechanism for the origin and evolution of life is in part dependent on its fidelity. We have devised a method for evaluating ligation fidelity in which ligation substrates are selected from random sequence libraries. The fidelities of chemical and enzymatic ligation are compared under a variety of conditions. While reaction conditions can be found that promote high fidelity copying, departure from these conditions leads to error-prone copying. In particular, ligation reactions with shorter oligonucleotide substrates are less efficient but more faithful. These results support a model for origins in which there was selective pressure for template-directed oligonucleotide ligation to be gradually supplanted by mononucleotide polymerization. },
  month = { Aug },
  pages = { 375--390 },
  number = { 4 },
  volume = { 29 },
  year = { 1999 },
  journal = { Orig Life Evol Biosph },
  title = { {T}he fidelity of template-directed oligonucleotide ligation and the inevitability of polymerase function },
  author = { James and Ellington },
}

@article{pmid10552046,
  abstract = { Molecular parasites that utilize the replication machinery of cells or of in vitro amplification reactions have previously been characterized. By and large, these parasites have been smaller than the viruses or amplicons that gave rise to them. This is likely because shorter genomes can be replicated more quickly. In contrast, we have identified and characterized parasites of an isothermal amplification reaction that are longer than their parental molecules yet replicate much more efficiently. These results raise interesting questions regarding whether the optimal size of replicators reflects a trade-off between the information encoded in a parasite and the information encoded in the machinery replicating that parasite. },
  month = { Nov },
  pages = { 656--663 },
  number = { 5 },
  volume = { 49 },
  year = { 1999 },
  journal = { J. Mol. Evol. },
  title = { {M}olecular parasites that evolve longer genomes },
  author = { Marshall and Ellington },
}

@article{pmid10542083,
  abstract = { Ribozymes that are sensitive to cyclic nucleotides have been selected in vitro. Remarkably, the cGMP-dependent ribozymes are specifically activated by a factor of 5,000 - the largest allosteric ribozyme activation by a small molecule seen to date. },
  month = { Nov },
  pages = { 992--994 },
  number = { 11 },
  volume = { 6 },
  year = { 1999 },
  journal = { Nat. Struct. Biol. },
  title = { {T}raining ribozymes to switch },
  author = { Marshall and Ellington },
}

% 27189550 
@Article{pmid27189550,
   Author="Hammerling, M. J.  and Gollihar, J.  and Mortensen, C.  and Alnahhas, R. N.  and Ellington, A. D.  and Barrick, J. E. ",
   Title="{{E}xpanded genetic codes create new mutational routes to rifampicin resistance in {E}scherichia coli}",
   Journal="Mol. Biol. Evol.",
   Year="2016",
   Pages=" ",
   Month="May",
   Abstract={Until recently, evolutionary questions surrounding the nature of the genetic code have been mostly limited to the realm of conjecture, modeling, and simulation due to the difficulty of altering this fundamental property of living organisms. Concerted genome and protein engineering efforts now make it possible to experimentally study the impact of alternative genetic codes on the evolution of biological systems. We explored how Escherichia coli strains that incorporate a 21(st) nonstandard amino acid (nsAA) at the recoded amber (TAG) stop codon evolve resistance to the antibiotic rifampicin. Resistance to rifampicin arises from chromosomal mutations in the β subunit of RNA polymerase (RpoB). We found that a variety of mutations that lead to substitutions of nsAAs in the essential RpoB protein confer robust rifampicin resistance. We interpret these results in a framework in which an expanded code can increase evolvability in two distinct ways: by adding a new letter with unique chemical properties to the protein alphabet and by altering the mutational connectivity of amber-adjacent codons by converting a lethal nonsense mutation into a missense mutation. Finally, we consider the implications of these results for the evolution of alternative genetic codes. In our experiments, reliance on a mutation to a reassigned codon for a vital trait is not required for the long-term maintenance of an expanded genetic code and may even destabilize incorporation of a nsAA, a result that is consistent with the codon capture model of genetic code evolution.}
}

% 27114511 
@Article{pmid27114511,
   Author="DeKosky, B. J.  and Lungu, O. I.  and Park, D.  and Johnson, E. L.  and Charab, W.  and Chrysostomou, C.  and Kuroda, D.  and Ellington, A. D.  and Ippolito, G. C.  and Gray, J. J.  and Georgiou, G. ",
   Title="{{L}arge-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2016",
   Volume="113",
   Number="19",
   Pages="E2636--2645",
   Month="May",
   Abstract={Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19(+)CD20(+)CD27(-) IgM-naive B cells, >120,000 antibody clusters from CD19(+)CD20(+)CD27(+) antigen-experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.}
}

% 27032410 
@Article{pmid27032410,
   Author="Jung, C.  and Ellington, A. D. ",
   Title="{{A} primerless molecular diagnostic: phosphorothioated-terminal hairpin formation and self-priming extension ({P}{S}-{T}{H}{S}{P})}",
   Journal="Anal Bioanal Chem",
   Year="2016",
   Pages=" ",
   Month="Mar",
   Abstract={There are various ways that priming can occur in nucleic acid amplification reactions. While most reactions rely on a primer to initiate amplification, a mechanism for DNA amplification has been developed in which hairpin sequences at the 3' terminus of a single-stranded oligonucleotide fold on themselves to initiate priming. Unfortunately, this method is less useful for diagnostic applications because the self-folding efficiency is low and only works over a narrow range of reaction temperatures. In order to adapt this strategy for analytical applications we have developed a variant that we term phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP). In PS-THSP a phosphorothioate (PS) modification is incorporated into the DNA backbone, leading to a reduction in the thermal stability of dsDNA and increased self-folding of terminal hairpins. By optimizing the number of PS linkages that are included in the initial template, we greatly increased self-folding efficiency and the range of reaction temperatures, ultimately achieving a detection limit of 1 pM. This improved method was readily adapted to the detection of single nucleotide polymorphisms and to the detection of non-nucleic acid analytes, such as alkaline phosphatase, which was quantitatively detected at a limit of 0.05 mU/mL, approximately 10-fold better than commercial assays. Graphical abstract Efficient self-folding by phosphorothioate (PS) modification.}
}

% 26899430 
@Article{pmid26899430,
   Author="Martini, L.  and Ellington, A. D.  and Mansy, S. S. ",
   Title="{{A}n in vitro selection for small molecule induced switching {R}{N}{A} molecules}",
   Journal="Methods",
   Year="2016",
   Pages=" ",
   Month="Feb",
   Abstract={The selection of RNA and DNA aptamers now has a long history. However, the ability to directly select for conformational changes upon ligand binding has remained elusive. These difficulties have stymied attempts at making small molecule responsive strand displacement circuitry as well as synthetic riboswitches. Herein we present a detailed strand displacement based selection protocol to directly select for RNA molecules with switching activity. The library was based on a previously selected thiamine pyrophosphate riboswitch. The fully in vitro methodology gave sequences that showed strong strand displacement activity in the presence of thiamine pyrophosphate. Further, the selected sequences possessed riboswitch activity similar to that of natural riboswitches. The presented methodology should aid in the design of more complex, environmentally responsive strand displacement circuitry and in the selection of riboswitches responsive to toxic ligands.}
}

% 26844430 
@Article{pmid26844430,
   Author="McDaniel, J. R.  and DeKosky, B. J.  and Tanno, H.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{U}ltra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes}",
   Journal="Nat Protoc",
   Year="2016",
   Volume="11",
   Number="3",
   Pages="429--442",
   Month="Mar",
   Abstract={High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >10(4) lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.}
}

% 26780407 
@Article{pmid26780407,
   Author="Tack, D. S.  and Ellefson, J. W.  and Thyer, R.  and Wang, B.  and Gollihar, J.  and Forster, M. T.  and Ellington, A. D. ",
   Title="{{A}ddicting diverse bacteria to a noncanonical amino acid}",
   Journal="Nat. Chem. Biol.",
   Year="2016",
   Volume="12",
   Number="3",
   Pages="138--140",
   Month="Mar",
   Abstract={Engineered orthogonal translation systems have greatly enabled the expansion of the genetic code using noncanonical amino acids (NCAAs). However, the impact of NCAAs on organismal evolution remains unclear, in part because it is difficult to force the adoption of new genetic codes in organisms. By reengineering TEM-1 β-lactamase to be dependent on a NCAA, we maintained bacterial NCAA dependence for hundreds of generations without escape.}
}

% 26753172 
@Article{pmid26753172,
   Author="Mahmood, M. A.  and Hasan, M. R.  and Khan, U. J.  and Allen, P. B.  and Kim, Y. T.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{O}ne-step tumor detection from dynamic morphology tracking on aptamer-grafted surfaces}",
   Journal="Technology (Singap World Sci)",
   Year="2015",
   Volume="3",
   Number="4",
   Pages="194--200",
   Month="Dec",
   Abstract={In this paper, we report a one-step tumor cell detection approach based on the dynamic morphological behavior tracking of cancer cells on a ligand modified surface. Every cell on the surface was tracked in real time for several minutes immediately after seeding until these were finally attached. Cancer cells were found to be very active in the aptamer microenvironment, changing their shapes rapidly from spherical to semi-elliptical, with much flatter spread and extending pseudopods at regular intervals. When incubated on a functionalized surface, the balancing forces between cell surface molecules and the surface-bound aptamers, together with the flexibility of the membranes, caused cells to show these distinct dynamic activities and variations in their morphologies. On the other hand, healthy cells remained distinguishingly inactive on the surface over the same period. The quantitative image analysis of cell morphologies provided feature vectors that were statistically distinct between normal and cancer cells.}
}

% 26750592 
@Article{pmid26750592,
   Author="Du, Y.  and Zhen, S. J.  and Li, B.  and Byrom, M.  and Jiang, Y. S.  and Ellington, A. D. ",
   Title="{{E}ngineering {S}ignaling {A}ptamers {T}hat {R}ely on {K}inetic {R}ather {T}han {E}quilibrium {C}ompetition}",
   Journal="Anal. Chem.",
   Year="2016",
   Volume="88",
   Number="4",
   Pages="2250--2257",
   Month="Feb",
   Abstract={During the past decade, aptasensors have largely been designed on the basis of the notion that ligand-modulated equilibration between aptamer conformations could be exploited for sensing. One implementation of this strategy has been to denature the aptamer with an antisense oligonucleotide, wait for dissociation of the antisense oligonucleotide, and stabilize the folded, signaling conformer with a ligand. However, there is a large kinetic barrier associated with releasing the oligonucleotide from the aptamer to again obtain an active, binding conformation. If the length of the antisense oligonucleotide is decreased to make dissociation from the aptamer more favorable, higher background signals are observed. To improve the general methodology for developing aptasensors, we have developed a novel and robust strategy for aptasensor design in which an oligonucleotide kinetically competes with the ligand for binding rather than having to be released from a stable duplex. While the oligonucleotide can induce conformational change, it initially chooses between the aptamer and a molecular beacon (MB), a process that does not require a lengthy pre-equilibration. Using an anti-ricin aptamer as a starting point, we developed a "competitive" aptasensor with a measured limit of detection (LOD) of 30 nM with an optical readout and as low as 3 nM for ricin toxin A-chain (RTA) detection on an electrochemical platform.}
}

% 26524397 
@Article{pmid26524397,
   Author="Jung, C.  and Allen, P. B.  and Ellington, A. D. ",
   Title="{{A} stochastic {D}{N}{A} walker that traverses a microparticle surface}",
   Journal="Nat Nanotechnol",
   Year="2016",
   Volume="11",
   Number="2",
   Pages="157--163",
   Month="Feb",
   Abstract={Molecular machines have previously been designed that are propelled by DNAzymes, protein enzymes and strand displacement. These engineered machines typically move along precisely defined one- and two-dimensional tracks. Here, we report a DNA walker that uses hybridization to drive walking on DNA-coated microparticle surfaces. Through purely DNA:DNA hybridization reactions, the nanoscale movements of the walker can lead to the generation of a single-stranded product and the subsequent immobilization of fluorescent labels on the microparticle surface. This suggests that the system could be of use in analytical and diagnostic applications, similar to how strand exchange reactions in solution have been used for transducing and quantifying signals from isothermal molecular amplification assays. The walking behaviour is robust and the walker can take more than 30 continuous steps. The traversal of an unprogrammed, inhomogeneous surface is also due entirely to autonomous decisions made by the walker, behaviour analogous to amorphous chemical reaction network computations, which have been shown to lead to pattern formation.}
}

% 26480829 
@Article{pmid26480829,
   Author="Ellington, A. D. ",
   Title="{{L}andscape-{B}ased {B}iology}",
   Journal="J. Mol. Evol.",
   Year="2015",
   Volume="81",
   Number="5-6",
   Pages="144--145",
   Month="Dec"
}

% 26471233 
@Article{pmid26471233,
   Author="Friedman, D. C.  and Ellington, A. D. ",
   Title="{{I}ndustrialization of {B}iology}",
   Journal="ACS Synth Biol",
   Year="2015",
   Volume="4",
   Number="10",
   Pages="1053--1055",
   Month="Oct",
   Abstract={The advancement of synthetic biology over the past decade has contributed substantially to the growing bioeconomy. A recent report by the National Academies highlighted several areas of advancement that will be needed for further expansion of industrial biotechnology, including new focuses on design, feedstocks, processing, organism development, and tools for testing and measurement; more particularly, a focus on expanded chassis and end-to-end design in an effort to move beyond the use of E. coli and S. cerivisiea to organisms better suited to fermentation and production; second, continued efforts in systems biology and high-throughput screening with a focus on more rapid techniques that will provide the needed information for moving to larger scale; and finally, work to accelerate the building of a holacratic community with collaboration and engagement between the relevant government agencies, industry, academia, and the public.}
}

% 26465114 
@Article{pmid26465114,
   Author="Quandt, E. M.  and Gollihar, J.  and Blount, Z. D.  and Ellington, A. D.  and Georgiou, G.  and Barrick, J. E. ",
   Title="{{F}ine-tuning citrate synthase flux potentiates and refines metabolic innovation in the {L}enski evolution experiment}",
   Journal="Elife",
   Year="2015",
   Volume="4",
   Pages=" ",
   Abstract={Evolutionary innovations that enable organisms to colonize new ecological niches are rare compared to gradual evolutionary changes in existing traits. We discovered that key mutations in the gltA gene, which encodes citrate synthase (CS), occurred both before and after Escherichia coli gained the ability to grow aerobically on citrate (Cit(+) phenotype) during the Lenski long-term evolution experiment. The first gltA mutation, which increases CS activity by disrupting NADH-inhibition of this enzyme, is beneficial for growth on the acetate and contributed to preserving the rudimentary Cit(+) trait from extinction when it first evolved. However, after Cit(+) was refined by further mutations, this potentiating gltA mutation became deleterious to fitness. A second wave of beneficial gltA mutations then evolved that reduced CS activity to below the ancestral level. Thus, dynamic reorganization of central metabolism made colonizing this new nutrient niche contingent on both co-opting and overcoming a history of prior adaptation.}
}

% 26355089 
@Article{pmid26355089,
   Author="Kerr, S. A.  and Jackson, E. L.  and Lungu, O. I.  and Meyer, A. G.  and Demogines, A.  and Ellington, A. D.  and Georgiou, G.  and Wilke, C. O.  and Sawyer, S. L. ",
   Title="{{C}omputational and {F}unctional {A}nalysis of the {V}irus-{R}eceptor {I}nterface {R}eveals {H}ost {R}ange {T}rade-{O}ffs in {N}ew {W}orld {A}renaviruses}",
   Journal="J. Virol.",
   Year="2015",
   Volume="89",
   Number="22",
   Pages="11643--11653",
   Month="Nov",
   Abstract={Animal viruses frequently cause zoonotic disease in humans. As these viruses are highly diverse, evaluating the threat that they pose remains a major challenge, and efficient approaches are needed to rapidly predict virus-host compatibility. Here, we develop a combined computational and experimental approach to assess the compatibility of New World arenaviruses, endemic in rodents, with the host TfR1 entry receptors of different potential new host species. Using signatures of positive selection, we identify a small motif on rodent TfR1 that conveys species specificity to the entry of viruses into cells. However, we show that mutations in this region affect the entry of each arenavirus differently. For example, a human single nucleotide polymorphism (SNP) in this region, L212V, makes human TfR1 a weaker receptor for one arenavirus, Machupo virus, but a stronger receptor for two other arenaviruses, Junin and Sabia viruses. Collectively, these findings set the stage for potential evolutionary trade-offs, where natural selection for resistance to one virus may make humans or rodents susceptible to other arenavirus species. Given the complexity of this host-virus interplay, we propose a computational method to predict these interactions, based on homology modeling and computational docking of the virus-receptor protein-protein interaction. We demonstrate the utility of this model for Machupo virus, for which a suitable cocrystal structural template exists. Our model effectively predicts whether the TfR1 receptors of different species will be functional receptors for Machupo virus entry. Approaches such at this could provide a first step toward computationally predicting the "host jumping" potential of a virus into a new host species.\\ We demonstrate how evolutionary trade-offs may exist in the dynamic evolutionary interplay between viruses and their hosts, where natural selection for resistance to one virus could make humans or rodents susceptible to other virus species. We present an algorithm that predicts which species have cell surface receptors that make them susceptible to Machupo virus, based on computational docking of protein structures. Few molecular models exist for predicting the risk of spillover of a particular animal virus into humans or new animal populations. Our results suggest that a combination of evolutionary analysis, structural modeling, and experimental verification may provide an efficient approach for screening and assessing the potential spillover risks of viruses circulating in animal populations.}
}

% 26355042 
@Article{pmid26355042,
   Author="Wang, B.  and Kluwe, C. A.  and Lungu, O. I.  and DeKosky, B. J.  and Kerr, S. A.  and Johnson, E. L.  and Jung, J.  and Rezigh, A. B.  and Carroll, S. M.  and Reyes, A. N.  and Bentz, J. R.  and Villanueva, I.  and Altman, A. L.  and Davey, R. A.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{F}acile {D}iscovery of a {D}iverse {P}anel of {A}nti-{E}bola {V}irus {A}ntibodies by {I}mmune {R}epertoire {M}ining}",
   Journal="Sci Rep",
   Year="2015",
   Volume="5",
   Pages="13926",
   Abstract={The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138(+) antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.}
}

% 26332362 
@Article{pmid26332362,
   Author="Mayfield, J. E.  and Fan, S.  and Wei, S.  and Zhang, M.  and Li, B.  and Ellington, A. D.  and Etzkorn, F. A.  and Zhang, Y. J. ",
   Title="{{C}hemical {T}ools {T}o {D}ecipher {R}egulation of {P}hosphatases by {P}roline {I}somerization on {E}ukaryotic {R}{N}{A} {P}olymerase {I}{I}}",
   Journal="ACS Chem. Biol.",
   Year="2015",
   Volume="10",
   Number="10",
   Pages="2405--2414",
   Month="Oct",
   Abstract={Proline isomerization greatly impacts biological signaling but is subtle and difficult to detect in proteins. We characterize this poorly understood regulatory mechanism for RNA polymerase II carboxyl terminal domain (CTD) phosphorylation state using novel, direct, and quantitative chemical tools. We determine the proline isomeric preference of three CTD phosphatases: Ssu72 as cis-proline specific, Scp1 and Fcp1 as strongly trans-preferred. Due to this inherent characteristic, these phosphatases respond differently to enzymes that catalyze the isomerization of proline, like Ess1/Pin1. We demonstrate that this selective regulation of RNA polymerase II phosphorylation state exists within human cells, consistent with in vitro assays. These results support a model in which, instead of a global enhancement of downstream enzymatic activities, proline isomerases selectively boost the activity of a subset of CTD regulatory factors specific for cis-proline. This leads to diversified phosphorylation states of CTD in vitro and in cells. We provide the chemical tools to investigate proline isomerization and its ability to selectively enhance signaling in transcription and other biological contexts.}
}

% 26224395 
@Article{pmid26224395,
   Author="Cunningham, J. C.  and Scida, K.  and Kogan, M. R.  and Wang, B.  and Ellington, A. D.  and Crooks, R. M. ",
   Title="{{P}aper diagnostic device for quantitative electrochemical detection of ricin at picomolar levels}",
   Journal="Lab Chip",
   Year="2015",
   Volume="15",
   Number="18",
   Pages="3707--3715",
   Abstract={We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min.}
}

% 26209133 
@Article{pmid26209133,
   Author="Meyer, A. J.  and Garry, D. J.  and Hall, B.  and Byrom, M. M.  and McDonald, H. G.  and Yang, X.  and Yin, Y. W.  and Ellington, A. D. ",
   Title="{{T}ranscription yield of fully 2'-modified {R}{N}{A} can be increased by the addition of thermostabilizing mutations to {T}7 {R}{N}{A} polymerase mutants}",
   Journal="Nucleic Acids Res.",
   Year="2015",
   Volume="43",
   Number="15",
   Pages="7480--7488",
   Month="Sep",
   Abstract={On average, mutations are deleterious to proteins. Mutations conferring new function to a protein often come at the expense of protein folding or stability, reducing overall activity. Over the years, a panel of T7 RNA polymerases have been designed or evolved to accept nucleotides with modified ribose moieties. These modified RNAs have proven useful, especially in vivo, but the transcriptional yields tend to be quite low. Here we show that mutations previously shown to increase the thermal tolerance of T7 RNA polymerase can increase the activity of mutants with expanded substrate range. The resulting polymerase mutants can be used to generate 2'-O-methyl modified RNA with yields much higher than enzymes currently employed.}
}

% 26201734 
@Article{pmid26201734,
   Author="Jung, C.  and Ellington, A. D. ",
   Title="{{S}ynthetic biology: {S}ix pack and stack}",
   Journal="Nat Chem",
   Year="2015",
   Volume="7",
   Number="8",
   Pages="617--619",
   Month="Aug"
}

% 26050646 
@Article{pmid26050646,
   Author="Du, Y.  and Hughes, R. A.  and Bhadra, S.  and Jiang, Y. S.  and Ellington, A. D.  and Li, B. ",
   Title="{{A} {S}weet {S}pot for {M}olecular {D}iagnostics: {C}oupling {I}sothermal {A}mplification and {S}trand {E}xchange {C}ircuits to {G}lucometers}",
   Journal="Sci Rep",
   Year="2015",
   Volume="5",
   Pages="11039",
   Abstract={Strand exchange nucleic acid circuitry can be used to transduce isothermal nucleic acid amplification products into signals that can be readable on an off-the-shelf glucometer. Loop-mediated isothermal amplification (LAMP) is limited by the accumulation of non-specific products, but nucleic acid circuitry can be used to probe and distinguish specific amplicons. By combining this high temperature isothermal amplification method with a thermostable invertase, we can directly transduce Middle-East respiratory syndrome coronavirus and Zaire Ebolavirus templates into glucose signals, with a sensitivity as low as 20-100 copies/μl, equating to atto-molar (or low zepto-mole). Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection. The method describes has potential for accelerating point-of-care applications, in that biological samples could be applied to a transducer that would then directly interface with an off-the-shelf, approved medical device.}
}

% 25984570 
@Article{pmid25984570,
   Author="Allen, P. B.  and Khaing, Z.  and Schmidt, C. E.  and Ellington, A. D. ",
   Title="{3{D} {P}rinting with {N}ucleic {A}cid {A}dhesives}",
   Journal="ACS Biomater Sci Eng",
   Year="2015",
   Volume="1",
   Number="1",
   Pages="19--26",
   Month="Jan",
   Abstract={By relying on specific DNA:DNA interactions as a "smart glue", we have assembled microparticles into a colloidal gel that can hold its shape. This gel can be extruded with a 3D printer to generate centimeter size objects. We show four aspects of this material: (1) The colloidal gel material holds its shape after extrusion. (2) The connectivity among the particles is controlled by the binding behavior between the surface DNA and this mediates some control over the microscale structure. (3) The use of DNA-coated microparticles dramatically reduces the cost of DNA-mediated assembly relative to conventional DNA nanotechnologies and makes this material accessible for macroscale applications. (4) This material can be assembled under biofriendly conditions and can host growing cells within its matrix. The DNA-based control over organization should provide a new means of engineering bioprinted tissues.}
}

% 25978303 
@Article{pmid25978303,
   Author="Martini, L.  and Meyer, A. J.  and Ellefson, J. W.  and Milligan, J. N.  and Forlin, M.  and Ellington, A. D.  and Mansy, S. S. ",
   Title="{{I}n {V}itro {S}election for {S}mall-{M}olecule-{T}riggered {S}trand {D}isplacement and {R}iboswitch {A}ctivity}",
   Journal="ACS Synth Biol",
   Year="2015",
   Volume="4",
   Number="10",
   Pages="1144--1150",
   Month="Oct",
   Abstract={An in vitro selection method for ligand-responsive RNA sensors was developed that exploited strand displacement reactions. The RNA library was based on the thiamine pyrophosphate (TPP) riboswitch, and RNA sequences capable of hybridizing to a target duplex DNA in a TPP regulated manner were identified. After three rounds of selection, RNA molecules that mediated a strand exchange reaction upon TPP binding were enriched. The enriched sequences also showed riboswitch activity. Our results demonstrated that small-molecule-responsive nucleic acid sensors can be selected to control the activity of target nucleic acid circuitry.}
}

% 25967118 
@Article{pmid25967118,
   Author="Milligan, J. N.  and Ellington, A. D. ",
   Title="{{U}sing {R}ec{A} protein to enhance kinetic rates of {D}{N}{A} circuits}",
   Journal="Chem. Commun. (Camb.)",
   Year="2015",
   Volume="51",
   Number="46",
   Pages="9503--9506",
   Month="Jun",
   Abstract={While DNA circuits are becoming increasingly useful as signal transducers, their utility is inhibited by their slow catalytic rate. Here, we demonstrate how RecA, a recombination enzyme that catalyzes sequence specific strand exchange, can be used to increase circuit rates up to 9-fold. We also show how the introduction of RNA into DNA circuits further controls the specificity of RecA strand exchange, improving signal-to-noise.}
}

% 25856093 
@Article{pmid25856093,
   Author="Bhadra, S.  and Jiang, Y. S.  and Kumar, M. R.  and Johnson, R. F.  and Hensley, L. E.  and Ellington, A. D. ",
   Title="{{R}eal-time sequence-validated loop-mediated isothermal amplification assays for detection of {M}iddle {E}ast respiratory syndrome coronavirus ({M}{E}{R}{S}-{C}o{V})}",
   Journal="PLoS ONE",
   Year="2015",
   Volume="10",
   Number="4",
   Pages="e0123126",
   Abstract={The Middle East respiratory syndrome coronavirus (MERS-CoV), an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF)1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD) for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU) (5 to 50 PFU/ml) of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.}
}

% 25826754 
@Article{pmid25826754,
   Author="Goodwin, S.  and Gade, A. M.  and Byrom, M.  and Herrera, B.  and Spears, C.  and Anslyn, E. V.  and Ellington, A. D. ",
   Title="{{N}ext-generation sequencing as input for chemometrics in differential sensing routines}",
   Journal="Angew. Chem. Int. Ed. Engl.",
   Year="2015",
   Volume="54",
   Number="21",
   Pages="6339--6342",
   Month="May",
   Abstract={Differential sensing (DS) methods traditionally use spatially arrayed receptors and optical signals to create score plots from multivariate data which classify individual analytes or complex mixtures. Herein, a new approach is described, in which nucleic acid sequences and sequence counts are used as the multivariate data without the necessity of a spatial array. To demonstrate this approach to DS, previously selected aptamers, identified from the literature, were used as semi-specific receptors, Next-Gen DNA sequencing was used to generate data, and cell line differentiation was the test-bed application. The study of a principal component analysis loading plot revealed cross-reactivity between the aptamers. The technique generates high-dimensionality score plots, and should be applicable to any mixture of complex and subtly different analytes for which nucleic acid-based receptors exist.}
}

% 25780159 
@Article{pmid25780159,
   Author="Ellington, A. D. ",
   Title="{{R}{N}{A} as a conception}",
   Journal="RNA",
   Year="2015",
   Volume="21",
   Number="4",
   Pages="608",
   Month="Apr"
}

% 25708458 
@Article{pmid25708458,
   Author="Jiang, Y. S.  and Bhadra, S.  and Li, B.  and Wu, Y. R.  and Milligan, J. N.  and Ellington, A. D. ",
   Title="{{R}obust strand exchange reactions for the sequence-specific, real-time detection of nucleic acid amplicons}",
   Journal="Anal. Chem.",
   Year="2015",
   Volume="87",
   Number="6",
   Pages="3314--3320",
   Month="Mar",
   Abstract={Loop-mediated isothermal amplification (LAMP) of DNA is a powerful isothermal nucleic acid amplification method that can generate upward of 10(9) copies from less than 100 copies of template DNA within an hour. Unfortunately, although the amplification reactions are extremely powerful, real-time and specific detection of LAMP products remains analytically challenging. In order to both improve the specificity of LAMP detection and to make readout simpler and more reliable, we have replaced the intercalating dye typically used for monitoring in real-time fluorescence with a toehold-mediated strand exchange reaction termed one-step strand displacement (OSD). Due to the inherent sequence specificity of toehold-mediated strand exchange, the OSD reporter could successfully distinguish side products from true amplicons arising from templates corresponding to the biomedically relevant M. tuberculosis RNA polymerase (rpoB) and the melanoma-related biomarker BRAF. OSD allowed the Yes/No detection of rpoB in a complex mixture such as synthetic sputum and also demonstrated single nucleotide specificity in Yes/No detection of a mutant BRAF allele (V600E) in the presence of 20-fold more of the wild-type gene. Real-time detection of different genes in multiplex LAMP reactions also proved possible. The development of simple, readily designed, modular equivalents of TaqMan probes for isothermal amplification reactions should generally improve the applicability of these reactions and may eventually assist with the development of point-of-care tests.}
}

% 25606981 
@Article{pmid25606981,
   Author="Stovall, G. M.  and Bedenbaugh, R. S.  and Singh, S.  and Meyer, A. J.  and Hatala, P. J.  and Ellington, A. D.  and Hall, B. ",
   Title="{{I}n vitro selection using modified or unnatural nucleotides}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2014",
   Volume="56",
   Pages="1--33",
   Abstract={Incorporation of modified nucleotides into in vitro RNA or DNA selections offers many potential advantages, such as the increased stability of selected nucleic acids against nuclease degradation, improved affinities, expanded chemical functionality, and increased library diversity. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for evaluating and optimizing transcription reactions, as well as confirming the incorporation of the modified nucleotides; protocols for evaluating modified nucleotide transcripts as template in reverse transcription reactions; protocols for the evaluation of the fidelity of modified nucleotides in the replication and the regeneration of the pool; and a protocol to compare modified nucleotide pools and selection conditions.}
}

% 25605388 
@Article{pmid25605388,
   Author="Bhadra, S.  and Ellington, A. D. ",
   Title="{{D}esign, synthesis, and application of {S}pinach molecular beacons triggered by strand displacement}",
   Journal="Meth. Enzymol.",
   Year="2015",
   Volume="550",
   Pages="215--249",
   Abstract={We describe design parameters for the synthesis and analytical application of a label-free RNA molecular beacon, termed Spinach.ST. The RNA aptamer Spinach fluoresces upon binding the small-molecule fluorophore DFHBI ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one). Spinach has been reengineered by extending its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation that fails to bind DHFBI. Hybridization of a trigger oligonucleotide to a designed toehold on Spinach.ST initiates toehold-mediated strand displacement and restores the DFHBI-binding, fluorescence-enhancing conformation of Spinach. The versatile Spinach.ST sensor can detect DNA or RNA trigger sequences and can readily distinguish single-nucleotide mismatches in the trigger toehold. Primer design techniques are described that augment amplicons produced by enzymatic amplification with Spinach.ST triggers. Interaction between these triggers and Spinach.ST molecular beacons leads to the real-time, sequence-specific quantitation of these amplicons. The use of Spinach.ST with isothermal amplification reactions such as nucleic acid sequence-based amplification (NASBA) may enable point-of-care applications. The same design principles could also be used to adapt Spinach reporters to the assay of nonnucleic acid analytes in trans.}
}

% 25603321 
@Article{pmid25603321,
   Author="P?osko?, E.  and Wagner, S. C.  and Ellington, A. D.  and Jewett, M. C.  and O'Reilly, R.  and Booth, P. J. ",
   Title="{{C}ontrolled assembly of artificial protein-protein complexes via {D}{N}{A} duplex formation}",
   Journal="Bioconjug. Chem.",
   Year="2015",
   Volume="26",
   Number="3",
   Pages="427--434",
   Month="Mar",
   Abstract={DNA-protein conjugates have found a wide range of applications. This study demonstrates the formation of defined, non-native protein-protein complexes via the site specific labeling of two proteins of interest with complementary strands of single-stranded DNA in vitro. This study demonstrates that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation.}
}

% 25521771 
@Article{pmid25521771,
   Author="Thyer, R.  and Robotham, S. A.  and Brodbelt, J. S.  and Ellington, A. D. ",
   Title="{{E}volving t{R}{N}{A}({S}ec) for efficient canonical incorporation of selenocysteine}",
   Journal="J. Am. Chem. Soc.",
   Year="2015",
   Volume="137",
   Number="1",
   Pages="46--49",
   Month="Jan",
   Abstract={Bacterial selenocysteine incorporation occurs in response to opal stop codons and is dependent on the presence of a selenocysteine insertion sequence (SECIS) element, which recruits the selenocysteine specific elongation factor and tRNA(Sec) needed to reassign the UGA codon. The SECIS element is a stem-loop RNA structure immediately following the UGA codon and forms part of the coding sequence in bacterial selenoproteins. Although the site specific incorporation of selenocysteine is of great interest for protein engineering, the sequence constraints imposed by the adjoining SECIS element severely limit its use. We have evolved an E. coli tRNA(Sec) that is compatible with the canonical translation machinery and can suppress amber stop codons to incorporate selenocysteine with high efficiency. This evolved tRNA(Sec) allows the production of new recombinant selenoproteins containing structural motifs such as selenyl-sulfhydryl and diselenide bonds.}
}

% 25506295 
@Article{pmid25506295,
   Author="Allen, P. B.  and Chen, X.  and Simpson, Z. B.  and Ellington, A. D. ",
   Title="{{M}odeling {S}calable {P}attern {G}eneration in {D}{N}{A} {R}eaction {N}etworks}",
   Journal="Nat Comput",
   Year="2014",
   Volume="13",
   Number="4",
   Pages="583--595",
   Month="Dec",
   Abstract={We have developed a theoretical framework for developing patterns in multiple dimensions using controllable diffusion and designed reactions implemented in DNA. This includes so-called strand displacement reactions in which one single-stranded DNA hybridizes to a hemi-duplex DNA and displaces another single-stranded DNA, reversibly or irreversibly. These reactions can be designed to proceed with designed rate and molecular specificity. By also controlling diffusion by partial complementarity to a stationary, cross-linked DNA, we can generate predictable patterns. We demonstrate this with several simulations showing deterministic, predictable shapes in space.}
}

% 25501908 
@Article{pmid25501908,
   Author="DeKosky, B. J.  and Kojima, T.  and Rodin, A.  and Charab, W.  and Ippolito, G. C.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{I}n-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire}",
   Journal="Nat. Med.",
   Year="2015",
   Volume="21",
   Number="1",
   Pages="86--91",
   Month="Jan",
   Abstract={High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or 'public', VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.}
}

% 25279711 
@Article{pmid25279711,
   Author="Meyer, A. J.  and Ellefson, J. W.  and Ellington, A. D. ",
   Title="{{D}irected {E}volution of a {P}anel of {O}rthogonal {T}7 {R}{N}{A} {P}olymerase {V}ariants for in {V}ivo or in {V}itro {S}ynthetic {C}ircuitry}",
   Journal="ACS Synth Biol",
   Year="2015",
   Volume="4",
   Number="10",
   Pages="1070--1076",
   Month="Oct",
   Abstract={T7 RNA polymerase is the foundation of synthetic biological circuitry both in vivo and in vitro due to its robust and specific control of transcription from its cognate promoter. Here we present the directed evolution of a panel of orthogonal T7 RNA polymerase:promoter pairs that each specifically recognizes a synthetic promoter. These newly described pairs can be used to independently control up to six circuits in parallel.}
}

% 25265557 
@Article{pmid25265557,
   Author="Enyeart, P. J.  and Simpson, Z. B.  and Ellington, A. D. ",
   Title="{{A} microbial model of economic trading and comparative advantage}",
   Journal="J. Theor. Biol.",
   Year="2015",
   Volume="364",
   Pages="326--343",
   Month="Jan",
   Abstract={The economic theory of comparative advantage postulates that beneficial trading relationships can be arrived at by two self-interested entities producing the same goods as long as they have opposing relative efficiencies in producing those goods. The theory predicts that upon entering trade, in order to maximize consumption both entities will specialize in producing the good they can produce at higher efficiency, that the weaker entity will specialize more completely than the stronger entity, and that both will be able to consume more goods as a result of trade than either would be able to alone. We extend this theory to the realm of unicellular organisms by developing mathematical models of genetic circuits that allow trading of a common good (specifically, signaling molecules) required for growth in bacteria in order to demonstrate comparative advantage interactions. In Conception 1, the experimenter controls production rates via exogenous inducers, allowing exploration of the parameter space of specialization. In Conception 2, the circuits self-regulate via feedback mechanisms. Our models indicate that these genetic circuits can demonstrate comparative advantage, and that cooperation in such a manner is particularly favored under stringent external conditions and when the cost of production is not overly high. Further work could involve implementing the models in living bacteria and searching for naturally occurring cooperative relationships between bacteria that conform to the principles of comparative advantage.}
}

% 25080493 
@Article{pmid25080493,
   Author="Segall-Shapiro, T. H.  and Meyer, A. J.  and Ellington, A. D.  and Sontag, E. D.  and Voigt, C. A. ",
   Title="{{A} 'resource allocator' for transcription based on a highly fragmented {T}7 {R}{N}{A} polymerase}",
   Journal="Mol. Syst. Biol.",
   Year="2014",
   Volume="10",
   Pages="742",
   Abstract={Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases (RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa 'σ fragment', and fragments with different specificity can direct the remaining 601-aa 'core fragment' to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal 'α fragment' and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids.}
}

% 25010201 
@Article{pmid25010201,
   Author="Du, Y.  and Lim, B. J.  and Li, B.  and Jiang, Y. S.  and Sessler, J. L.  and Ellington, A. D. ",
   Title="{{R}eagentless, ratiometric electrochemical {D}{N}{A} sensors with improved robustness and reproducibility}",
   Journal="Anal. Chem.",
   Year="2014",
   Volume="86",
   Number="15",
   Pages="8010--8016",
   Month="Aug",
   Abstract={To make the electrochemical DNA sensors (E-sensor) more robust and reproducible, we have now for the first time adapted the techniques of ratiometric analyses to the field of E-sensors. We did this via the simple expedient way of simultaneously using two redox probes: Methylene blue as the reporter of the conformational change, and ferrocene as an internal control. During the conformational transduction, only the distance between the signal probe and the electrode surface undergoes an appreciable change, while the distance between the control probe and the electrode remains relatively constant. This special design has allowed very reliable target recognition, as illustrated in this report using a human T-lymphotropic virus type I gene fragment. The standard deviation between measurements obtained using different electrodes was an order of magnitude less than that obtained using a classic E-sensor, which we prepared as a control. A limit of detection of 25.1 pM was obtained with our new system, with a single mismatch discrimination factor of 2.33 likewise being observed. Additionally, this concept had general applicability, and preliminary data of a "Signal-On" ratiometric E-sensor are also provided. Taken in concert, these results serve to validate the utility of what we believe will emerge as an easily generalized approach to oligonucleotide recognition and sensing.}
}

% 24990378 
@Article{pmid24990378,
   Author="Byrom, M.  and Bhadra, S.  and Jiang, Y. S.  and Ellington, A. D. ",
   Title="{{E}xquisite allele discrimination by toehold hairpin primers}",
   Journal="Nucleic Acids Res.",
   Year="2014",
   Volume="42",
   Number="15",
   Pages="e120",
   Month="Sep",
   Abstract={The ability to detect and monitor single nucleotide polymorphisms (SNPs) in biological samples is an enabling research and clinical tool. We have developed a surprising, inexpensive primer design method that provides exquisite discrimination between SNPs. The field of DNA computation is largely reliant on using so-called toeholds to initiate strand displacement reactions, leading to the execution of kinetically trapped circuits. We have now similarly found that the short toehold sequence to a target of interest can initiate both strand displacement within the hairpin and extension of the primer by a polymerase, both of which will further stabilize the primer:template complex. However, if the short toehold does not bind, neither of these events can readily occur and thus amplification should not occur. Toehold hairpin primers were used to detect drug resistance alleles in two genes, rpoB and katG, in the Mycobacterium tuberculosis genome, and ten alleles in the Escherichia coli genome. During real-time PCR, the primers discriminate between mismatched templates with Cq delays that are frequently so large that the presence or absence of mismatches is essentially a 'yes/no' answer.}
}

% 24942625 
@Article{pmid24942625,
   Author="Bhadra, S.  and Ellington, A. D. ",
   Title="{{A} {S}pinach molecular beacon triggered by strand displacement}",
   Journal="RNA",
   Year="2014",
   Volume="20",
   Number="8",
   Pages="1183--1194",
   Month="Aug",
   Abstract={We have re-engineered the fluorescent RNA aptamer Spinach to be activated in a sequence-dependent manner. The original Spinach aptamer was extended at its 5'- and 3'-ends to create Spinach.ST, which is predicted to fold into an inactive conformation and thus prevent association with the small molecule fluorophore DFHBI. Hybridization of a specific trigger oligonucleotide to a designed toehold leads to toehold-initiated strand displacement and refolds Spinach into the active, fluorophore-binding conformation. Spinach.ST not only specifically detects its target oligonucleotide but can discriminate readily against single-nucleotide mismatches. RNA amplicons produced during nucleic acid sequence-based amplification (NASBA) of DNA or RNA targets could be specifically detected and reported in real-time by conformational activation of Spinach.ST generated by in vitro transcription. In order to adapt any target sequence to detection by a Spinach reporter we used a primer design technique that brings together otherwise distal toehold sequences via hairpin formation. The same techniques could potentially be used to adapt common Spinach reporters to non-nucleic acid analytes, rather than by making fusions between aptamers and Spinach.}
}

% 24828239 
@Article{pmid24828239,
   Author="Jung, C.  and Ellington, A. D. ",
   Title="{{D}iagnostic applications of nucleic acid circuits}",
   Journal="Acc. Chem. Res.",
   Year="2014",
   Volume="47",
   Number="6",
   Pages="1825--1835",
   Month="Jun",
   Abstract={CONSPECTUS: While the field of DNA computing and molecular programming was engendered in large measure as a curiosity-driven exercise, it has taken on increasing importance for analytical applications. This is in large measure because of the modularity of DNA circuitry, which can serve as a programmable intermediate between inputs and outputs. These qualities may make nucleic acid circuits useful for making decisions relevant to diagnostic applications. This is especially true given that nucleic acid circuits can potentially directly interact with and be triggered by diagnostic nucleic acids and other analytes. Chemists are, by and large, unaware of many of these advances, and this Account provides a means of touching on what might seem to be an arcane field. We begin by explaining nucleic acid amplification reactions that can lead to signal amplification, such as catalytic hairpin assembly (CHA) and the hybridization chain reaction (HCR). In these circuits, a single-stranded input acts on kinetically trapped substrates via exposed toeholds and strand exchange reactions, refolding the substrates and allowing them to interact with one another. As multiple duplexes (CHA) or concatemers of increasing length (HCR) are generated, there are opportunities to couple these outputs to different analytical modalities, including transduction to fluorescent, electrochemical, and colorimetric signals. Because both amplification and transduction are at their root dependent on the programmability of Waston-Crick base pairing, nucleic acid circuits can be much more readily tuned and adapted to new applications than can many other biomolecular amplifiers. As an example, robust methods for real-time monitoring of isothermal amplification reactions have been developed recently. Beyond amplification, nucleic acid circuits can include logic gates and thresholding components that allow them to be used for analysis and decision making. Scalable and complex DNA circuits (seesaw gates) capable of carrying out operations such as taking square roots or implementing neural networks capable of learning have now been constructed. Into the future, we can expect that molecular circuitry will be designed to make decisions on the fly that reconfigure diagnostic devices or lead to new treatment options.}
}

% 24754567 
@Article{pmid24754567,
   Author="Melancon, M. P.  and Zhou, M.  and Zhang, R.  and Xiong, C.  and Allen, P.  and Wen, X.  and Huang, Q.  and Wallace, M.  and Myers, J. N.  and Stafford, R. J.  and Liang, D.  and Ellington, A. D.  and Li, C. ",
   Title="{{S}elective uptake and imaging of aptamer- and antibody-conjugated hollow nanospheres targeted to epidermal growth factor receptors overexpressed in head and neck cancer}",
   Journal="ACS Nano",
   Year="2014",
   Volume="8",
   Number="5",
   Pages="4530--4538",
   Month="May",
   Abstract={The purpose of this study was to compare the binding affinity and selective targeting of aptamer- and antibody-coated hollow gold nanospheres (HAuNS) targeted to epidermal growth factor receptors (EGFR). EGFR-targeting aptamers were conjugated to HAuNS (apt-HAuNS) by attaching a thiol-terminated single-stranded DNA to the HAuNS and then adding the complementary RNA targeted to EGFR. Apt-HAuNS was characterized in terms of size, surface charge, absorption, and number of aptamers per particle. The in vivo pharmacokinetics, in vivo biodistribution, and micro-SPECT/CT imaging of (111)In-labeled apt-HAuNS and anti-EGFR antibody (C225)-conjugated HAuNS were evaluated in nude mice bearing highly malignant human OSC-19 oral tumors. (111)In-labeled PEG-HAuNS was used as a control (n = 5/group). Apt-HAuNS did not have an altered absorbance profile or size (λmax = 800 nm; diameter = 55 nm) compared to C225-HAuNS or PEG-HAuNS. The surface charge became more negative upon conjugation of the aptamer (-51.4 vs -19.0 for PEG-HAuNS and -25.0 for C225-HAuNS). The number of aptamers/particle was ∼250. In vitro cell binding and in vivo biodistribution showed selective binding of the apt-HAuNS to EGFR. μSPECT/CT imaging confirmed that there was more tumor uptake of apt-HAuNS than C225-HAuNS. Aptamer is a promising ligand for image-guided delivery of nanoparticles for treatment of tumor cells overexpressing EGFR.}
}

% 24631721 
@Article{pmid24631721,
   Author="Singh-Blom, A.  and Hughes, R. A.  and Ellington, A. D. ",
   Title="{{A}n amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins}",
   Journal="J. Biotechnol.",
   Year="2014",
   Volume="178",
   Pages="12--22",
   Month="May",
   Abstract={Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.}
}

% 24625092 
@Article{pmid24625092,
   Author="Lu, W. C.  and Ellington, A. D. ",
   Title="{{D}esign and selection of a synthetic operon}",
   Journal="ACS Synth Biol",
   Year="2014",
   Volume="3",
   Number="6",
   Pages="410--415",
   Month="Jun",
   Abstract={Cell-free systems are showing increasing promise for biosynthesis of both proteins and small molecules. However, in vitro transcription and translation reactions have so far primarily been used for the production of single proteins. In order to demonstrate the possibilities for coupled reactions, we designed synthetic operons that included different combinations of wild-type or evolved biotin ligases and streptavidins and demonstrated a mechanism for self-selection of operons following expression in vitro. Peptide substrates for biotin ligase were conjugated to the DNA operons and could be modified by a biotin ligase specific for either biotin or desthiobiotin and subsequently captured via a streptavidin specific for either biotin or desthiobiotin.}
}

% 24624315 
@Article{pmid24624315,
   Author="Meyer, A. G.  and Sawyer, S. L.  and Ellington, A. D.  and Wilke, C. O. ",
   Title="{{A}nalyzing machupo virus-receptor binding by molecular dynamics simulations}",
   Journal="PeerJ",
   Year="2014",
   Volume="2",
   Pages="e266",
   Abstract={In many biological applications, we would like to be able to computationally predict mutational effects on affinity in protein-protein interactions. However, many commonly used methods to predict these effects perform poorly in important test cases. In particular, the effects of multiple mutations, non alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here an existing method applied in a novel way to a new test case; we interrogate affinity differences resulting from mutations in a host-virus protein-protein interface. We use steered molecular dynamics (SMD) to computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1). We then approximate affinity using the maximum applied force of separation and the area under the force-versus-distance curve. We find, even without the rigor and planning required for free energy calculations, that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Moreover, we show that this simple SMD scheme correlates well with relative free energy differences computed via free energy perturbation. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our approach provides a framework to compare the effects of multiple mutations, individually and jointly, on protein-protein interactions.}
}

% 24510437 
@Article{pmid24510437,
   Author="Meyer, A. J.  and Ellefson, J. W.  and Ellington, A. D. ",
   Title="{{L}ibrary generation by gene shuffling}",
   Journal="Curr Protoc Mol Biol",
   Year="2014",
   Volume="105",
   Pages="Unit 15.12.",
   Abstract={This unit describes the process of gene shuffling, also known as sexual PCR. Gene shuffling is a facile method for the generation of sequence libraries containing the information from a family of related genes. Essentially, related genes are fragmented by DNase I digestion and reassembled by primer-less PCR. The resulting chimeric genes can then be screened or selected for a desired function.}
}

% 24493736 
@Article{pmid24493736,
   Author="Bhadra, S.  and Ellington, A. D. ",
   Title="{{D}esign and application of cotranscriptional non-enzymatic {R}{N}{A} circuits and signal transducers}",
   Journal="Nucleic Acids Res.",
   Year="2014",
   Volume="42",
   Number="7",
   Pages="e58",
   Month="Apr",
   Abstract={Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer 'Spinach' that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits.}
}

% 24488121 
@Article{pmid24488121,
   Author="O'Connell, J. D.  and Tsechansky, M.  and Royall, A.  and Boutz, D. R.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{A} proteomic survey of widespread protein aggregation in yeast}",
   Journal="Mol Biosyst",
   Year="2014",
   Volume="10",
   Number="4",
   Pages="851--861",
   Month="Apr",
   Abstract={Many normally cytosolic yeast proteins form insoluble intracellular bodies in response to nutrient depletion, suggesting the potential for widespread protein aggregation in stressed cells. Nearly 200 such bodies have been found in yeast by screening libraries of fluorescently tagged proteins. In order to more broadly characterize the formation of these bodies in response to stress, we employed a proteome-wide shotgun mass spectrometry assay in order to measure shifts in the intracellular solubilities of endogenous proteins following heat stress. As quantified by mass spectrometry, heat stress tended to shift the same proteins into insoluble form as did nutrient depletion; many of these proteins were also known to form foci in response to arsenic stress. Affinity purification of several foci-forming proteins showed enrichment for co-purifying chaperones, including Hsp90 chaperones. Tests of induction conditions and co-localization of metabolic enzymes participating in the same metabolic pathways suggested those foci did not correspond to multi-enzyme organizing centers. Thus, in yeast, the formation of stress bodies appears common across diverse, normally diffuse cytoplasmic proteins and is induced by multiple types of cell stress, including thermal, chemical, and nutrient stress.}
}

% 24487692 
@Article{pmid24487692,
   Author="Hammerling, M. J.  and Ellefson, J. W.  and Boutz, D. R.  and Marcotte, E. M.  and Ellington, A. D.  and Barrick, J. E. ",
   Title="{{B}acteriophages use an expanded genetic code on evolutionary paths to higher fitness}",
   Journal="Nat. Chem. Biol.",
   Year="2014",
   Volume="10",
   Number="3",
   Pages="178--180",
   Month="Mar",
   Abstract={Bioengineering advances have made it possible to fundamentally alter the genetic codes of organisms. However, the evolutionary consequences of expanding an organism's genetic code with a noncanonical amino acid are poorly understood. Here we show that bacteriophages evolved on a host that incorporates 3-iodotyrosine at the amber stop codon acquire neutral and beneficial mutations to this new amino acid in their proteins, demonstrating that an expanded genetic code increases evolvability.}
}

% 24469811 
@Article{pmid24469811,
   Author="Lavinder, J. J.  and Wine, Y.  and Giesecke, C.  and Ippolito, G. C.  and Horton, A. P.  and Lungu, O. I.  and Hoi, K. H.  and DeKosky, B. J.  and Murrin, E. M.  and Wirth, M. M.  and Ellington, A. D.  and Dorner, T.  and Marcotte, E. M.  and Boutz, D. R.  and Georgiou, G. ",
   Title="{{I}dentification and characterization of the constituent human serum antibodies elicited by vaccination}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2014",
   Volume="111",
   Number="6",
   Pages="2259--2264",
   Month="Feb",
   Abstract={Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT(+) serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with Kd ∼ 10(-8)-10(-10) M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3(-)CD14(-)CD19(+)CD27(++)CD38(++)CD20(-)TT(+)) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.}
}

% 24436411 
@Article{pmid24436411,
   Author="Gollihar, J.  and Levy, M.  and Ellington, A. D. ",
   Title="{{B}iochemistry. {M}any paths to the origin of life}",
   Journal="Science",
   Year="2014",
   Volume="343",
   Number="6168",
   Pages="259--260",
   Month="Jan"
}

% 24413256 
@Article{pmid24413256,
   Author="Zhao, A.  and Tsechansky, M.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{R}evisiting and revising the purinosome}",
   Journal="Mol Biosyst",
   Year="2014",
   Volume="10",
   Number="3",
   Pages="369--374",
   Month="Mar",
   Abstract={Some metabolic pathway enzymes are known to organize into multi-enzyme complexes for reasons of catalytic efficiency, metabolite channeling, and other advantages of compartmentalization. It has long been an appealing prospect that de novo purine biosynthesis enzymes form such a complex, termed the "purinosome." Early work characterizing these enzymes garnered scarce but encouraging evidence for its existence. Recent investigations led to the discovery in human cell lines of purinosome bodies-cytoplasmic puncta containing transfected purine biosynthesis enzymes, which were argued to correspond to purinosomes. New discoveries challenge both the functional and physiological relevance of these bodies in favor of protein aggregation.}
}

% 24410776 
@Article{pmid24410776,
   Author="Enyeart, P. J.  and Mohr, G.  and Ellington, A. D.  and Lambowitz, A. M. ",
   Title="{{B}iotechnological applications of mobile group {I}{I} introns and their reverse transcriptases: gene targeting, {R}{N}{A}-seq, and non-coding {R}{N}{A} analysis}",
   Journal="Mob DNA",
   Year="2014",
   Volume="5",
   Number="1",
   Pages="2",
   Abstract={Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into 'targetrons.' Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and 'cut-and-pastes' (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.}
}

% 24402831 
@Article{pmid24402831,
   Author="Jiang, Y. S.  and Bhadra, S.  and Li, B.  and Ellington, A. D. ",
   Title="{{M}ismatches improve the performance of strand-displacement nucleic {A}cid circuits}",
   Journal="Angew. Chem. Int. Ed. Engl.",
   Year="2014",
   Volume="53",
   Number="7",
   Pages="1845--1848",
   Month="Feb",
   Abstract={Catalytic hairpin assembly (CHA) has previously proven useful as a transduction and amplification method for nucleic acid detection. However, the two hairpin substrates in a CHA circuit can potentially react non-specifically even in the absence of a single-stranded catalyst, and this non-specific background degrades the signal-to-noise ratio. The introduction of mismatched base pairs that impede uncatalyzed strand exchange reactions led to a significant decrease of the background signal, while only partially damping the signal in the presence of a catalyst. Various types and lengths of mismatches were assayed by fluorimetry, and in many instances, our MismatCHA designs yielded 100-fold increased signal-to-background ratios compared to a ratio of 4:1 with the perfectly matched substrates. These observations could be of general utility for the design of non-enzymatic nucleic acid circuits.}
}

% 24379390 
@Article{pmid24379390,
   Author="Quandt, E. M.  and Deatherage, D. E.  and Ellington, A. D.  and Georgiou, G.  and Barrick, J. E. ",
   Title="{{R}ecursive genomewide recombination and sequencing reveals a key refinement step in the evolution of a metabolic innovation in {E}scherichia coli}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2014",
   Volume="111",
   Number="6",
   Pages="2217--2222",
   Month="Feb",
   Abstract={Evolutionary innovations often arise from complex genetic and ecological interactions, which can make it challenging to understand retrospectively how a novel trait arose. In a long-term experiment, Escherichia coli gained the ability to use abundant citrate (Cit(+)) in the growth medium after ∼31,500 generations of evolution. Exploiting this previously untapped resource was highly beneficial: later Cit(+) variants achieve a much higher population density in this environment. All Cit(+) individuals share a mutation that activates aerobic expression of the citT citrate transporter, but this mutation confers only an extremely weak Cit(+) phenotype on its own. To determine which of the other >70 mutations in early Cit(+) clones were needed to take full advantage of citrate, we developed a recursive genomewide recombination and sequencing method (REGRES) and performed genetic backcrosses to purge mutations not required for Cit(+) from an evolved strain. We discovered a mutation that increased expression of the dctA C4-dicarboxylate transporter greatly enhanced the Cit(+) phenotype after it evolved. Surprisingly, strains containing just the citT and dctA mutations fully use citrate, indicating that earlier mutations thought to have potentiated the initial evolution of Cit(+) are not required for expression of the refined version of this trait. Instead, this metabolic innovation may be contingent on a genetic background, and possibly ecological context, that enabled citT mutants to persist among competitors long enough to obtain dctA or equivalent mutations that conferred an overwhelming advantage. More generally, refinement of an emergent trait from a rudimentary form may be crucial to its evolutionary success.}
}

% 24375025 
@Article{pmid24375025,
   Author="Lu, W. C.  and Levy, M.  and Kincaid, R.  and Ellington, A. D. ",
   Title="{{D}irected evolution of the substrate specificity of biotin ligase}",
   Journal="Biotechnol. Bioeng.",
   Year="2014",
   Volume="111",
   Number="6",
   Pages="1071--1081",
   Month="Jun",
   Abstract={We have developed selection scheme for directing the evolution of Escherichia coli biotin protein ligase (BPL) via in vitro compartmentalization, and have used this scheme to alter the substrate specificity of the ligase towards the utilization of the biotin analogue desthiobiotin. In this scheme, a peptide substrate (BAP) was conjugated to a DNA library encoding BirA, emulsified such that there was a single template per compartment, and protein variants were transcribed and translated in vitro. Those variants that could efficiently desthiobiotinylate their corresponding peptide:DNA conjugate were subsequently captured and amplified. Following just six rounds of selection and amplification several variants that demonstrated higher activity with desthiobiotin were identified. The best variants from Round 6, BirA6-40 and BirA6-47 , showed 17-fold and 10-fold higher activity, respectively, their abilities to use desthiobiotin as a substrate. While selected enzymes contained a number of substitutions, a single mutation, M157T, proved sufficient to provide much greater activity with desthiobiotin. Further characterization of BirA6-40 and the single substitution variant BirAM157T revealed that they had twoto threefold higher kcat values for desthiobiotin. These variants had also lost much of their ability to utilize biotin, resulting in orthogonal enzymes that in conjunction with streptavidin variants that can utilize desthiobiotin may prove to be of great use in developing additional, robust conjugation handles for a variety of biological and biotechnological applications.}
}

% 24256862 
@Article{pmid24256862,
   Author="Chirieleison, S. M.  and Allen, P. B.  and Simpson, Z. B.  and Ellington, A. D.  and Chen, X. ",
   Title="{{P}attern transformation with {D}{N}{A} circuits}",
   Journal="Nat Chem",
   Year="2013",
   Volume="5",
   Number="12",
   Pages="1000--1005",
   Month="Dec",
   Abstract={Readily programmable chemical networks are important tools as the scope of chemistry expands from individual molecules to larger molecular systems. Although many complex systems are constructed using conventional organic and inorganic chemistry, the programmability of biological molecules such as nucleic acids allows for precise, high-throughput and automated design, as well as simple, rapid and robust implementation. Here we show that systematic and quantitative control over the diffusivity and reactivity of DNA molecules yields highly programmable chemical reaction networks (CRNs) that execute at the macroscale. In particular, we designed and implemented non-enzymatic DNA circuits capable of performing pattern-transformation algorithms such as edge detection. We also showed that it is possible to fine-tune and multiplex such circuits. We believe these strategies will provide programmable platforms on which to prototype CRNs, discover bottom-up construction principles and generate patterns in materials.}
}

% 24214216 
@Article{pmid24214216,
   Author="Ledbetter, M. P.  and Hwang, T. W.  and Stovall, G. M.  and Ellington, A. D. ",
   Title="{{C}ontinuous in vitro evolution of a ribozyme ligase: a model experiment for the evolution of a biomolecule}",
   Journal="Biochem Mol Biol Educ",
   Year="2013",
   Volume="41",
   Number="6",
   Pages="433--442",
   Abstract={Evolution is a defining criterion of life and is central to understanding biological systems. However, the timescale of evolutionary shifts in phenotype limits most classroom evolution experiments to simple probability simulations. In vitro directed evolution (IVDE) frequently serves as a model system for the study of Darwinian evolution but produces noticeable phenotypic shifts in a matter of hours. An IVDE demonstration lab would serve to both directly demonstrate how Darwinian selection can act on a pool of variants and introduce students to an essential method of modern molecular biology. To produce an IVDE demonstration lab, continuous IVDE of a T500 ribozyme ligase population has been paired with a fluorescent strand displacement reporter system to visualize the selection of improved catalytic function. A ribozyme population is taken through rounds of isothermal amplification dependent on the self-ligation of a T7 promoter. As the population is selectively enriched with better ligase activity, the strand displacement system allows for the monitoring of the population's ligation rate. The strand displacement reporter system permits the detection of ligated ribozyme. Once ligated with the T7 promoter, the 5' end of the ribozyme displaces paired fluorophore-quencher oligonucleotides, in turn, generating visible signal upon UV light excitation. As the ligation rate of the population increases, due to the selection for faster ligating species, the fluorescent signal develops more rapidly. The pairing of the continuous isothermal system with the fluorescent reporting scheme allows any user, provided with minimal materials, to model the continuous directed evolution of a biomolecule.}
}

% 24185096 
@Article{pmid24185096,
   Author="Ellefson, J. W.  and Meyer, A. J.  and Hughes, R. A.  and Cannon, J. R.  and Brodbelt, J. S.  and Ellington, A. D. ",
   Title="{{D}irected evolution of genetic parts and circuits by compartmentalized partnered replication}",
   Journal="Nat. Biotechnol.",
   Year="2014",
   Volume="32",
   Number="1",
   Pages="97--101",
   Month="Jan",
   Abstract={Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.}
}

% 24135653 
@Article{pmid24135653,
   Author="Bhadra, S.  and Codrea, V.  and Ellington, A. D. ",
   Title="{{G}-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons}",
   Journal="Anal. Biochem.",
   Year="2014",
   Volume="445",
   Pages="38--40",
   Month="Jan",
   Abstract={We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5'-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.}
}

% 24070108 
@Article{pmid24070108,
   Author="Scida, K.  and Li, B.  and Ellington, A. D.  and Crooks, R. M. ",
   Title="{{D}{N}{A} detection using origami paper analytical devices}",
   Journal="Anal. Chem.",
   Year="2013",
   Volume="85",
   Number="20",
   Pages="9713--9720",
   Month="Oct",
   Abstract={We demonstrate the hybridization-induced fluorescence detection of DNA on an origami-based paper analytical device (oPAD). The paper substrate was patterned by wax printing and controlled heating to construct hydrophilic channels and hydrophobic barriers in a three-dimensional fashion. A competitive assay was developed where the analyte, a single-stranded DNA (ssDNA), and a quencher-labeled ssDNA competed for hybridization with a fluorophore-labeled ssDNA probe. Upon hybridization of the analyte with the fluorophore-labeled ssDNA, a linear response of fluorescence vs analyte concentration was observed with an extrapolated limit of detection <5 nM and a sensitivity relative standard deviation as low as 3%. The oPAD setup was also tested against OR/AND logic gates, proving to be successful in both detection systems.}
}

% 24002656 
@Article{pmid24002656,
   Author="Enyeart, P. J.  and Chirieleison, S. M.  and Dao, M. N.  and Perutka, J.  and Quandt, E. M.  and Yao, J.  and Whitt, J. T.  and Keatinge-Clay, A. T.  and Lambowitz, A. M.  and Ellington, A. D. ",
   Title="{{G}eneralized bacterial genome editing using mobile group {I}{I} introns and {C}re-lox}",
   Journal="Mol. Syst. Biol.",
   Year="2013",
   Volume="9",
   Pages="685",
   Abstract={Efficient bacterial genetic engineering approaches with broad-host applicability are rare. We combine two systems, mobile group II introns ('targetrons') and Cre/lox, which function efficiently in many different organisms, into a versatile platform we call GETR (Genome Editing via Targetrons and Recombinases). The introns deliver lox sites to specific genomic loci, enabling genomic manipulations. Efficiency is enhanced by adding flexibility to the RNA hairpins formed by the lox sites. We use the system for insertions, deletions, inversions, and one-step cut-and-paste operations. We demonstrate insertion of a 12-kb polyketide synthase operon into the lacZ gene of Escherichia coli, multiple simultaneous and sequential deletions of up to 120 kb in E. coli and Staphylococcus aureus, inversions of up to 1.2 Mb in E. coli and Bacillus subtilis, and one-step cut-and-pastes for translocating 120 kb of genomic sequence to a site 1.5 Mb away. We also demonstrate the simultaneous delivery of lox sites into multiple loci in the Shewanella oneidensis genome. No selectable markers need to be placed in the genome, and the efficiency of Cre-mediated manipulations typically approaches 100%.}
}

% 23844594 
@Article{pmid23844594,
   Author="Luo, Y.  and Yogesha, S. D.  and Cannon, J. R.  and Yan, W.  and Ellington, A. D.  and Brodbelt, J. S.  and Zhang, Y. ",
   Title="{novel modifications on {C}-terminal domain of {R}{N}{A} polymerase {I}{I} can fine-tune the phosphatase activity of {S}su72}",
   Journal="ACS Chem. Biol.",
   Year="2013",
   Volume="8",
   Number="9",
   Pages="2042--2052",
   Month="Sep",
   Abstract={The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats, through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intramolecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 does not dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 toward the CTD peptide by 4-fold. To our surprise, even though the intramolecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.}
}

% 23754529 
@Article{pmid23754529,
   Author="Wilson, R. H.  and Morton, S. K.  and Deiderick, H.  and Gerth, M. L.  and Paul, H. A.  and Gerber, I.  and Patel, A.  and Ellington, A. D.  and Hunicke-Smith, S. P.  and Patrick, W. M. ",
   Title="{{E}ngineered {D}{N}{A} ligases with improved activities in vitro}",
   Journal="Protein Eng. Des. Sel.",
   Year="2013",
   Volume="26",
   Number="7",
   Pages="471--478",
   Month="Jul",
   Abstract={The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.}
}

% 23741319 
@Article{pmid23741319,
   Author="Der, B. S.  and Kluwe, C.  and Miklos, A. E.  and Jacak, R.  and Lyskov, S.  and Gray, J. J.  and Georgiou, G.  and Ellington, A. D.  and Kuhlman, B. ",
   Title="{{A}lternative computational protocols for supercharging protein surfaces for reversible unfolding and retention of stability}",
   Journal="PLoS ONE",
   Year="2013",
   Volume="8",
   Number="5",
   Pages="e64363",
   Abstract={Reengineering protein surfaces to exhibit high net charge, referred to as "supercharging", can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from -11 to -61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and -30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.}
}

% 23680795 
@Article{pmid23680795,
   Author="Xu, J.  and Tack, D.  and Hughes, R. A.  and Ellington, A. D.  and Gray, J. J. ",
   Title="{{S}tructure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex}",
   Journal="J. Struct. Biol.",
   Year="2014",
   Volume="185",
   Number="2",
   Pages="215--222",
   Month="Feb",
   Abstract={Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.}
}

% 23657394 
@Article{pmid23657394,
   Author="Maranhao, A. C.  and Ellington, A. D. ",
   Title="{{E}ndowing cells with logic and memory}",
   Journal="Nat. Biotechnol.",
   Year="2013",
   Volume="31",
   Number="5",
   Pages="413--415",
   Month="May"
}

% 23651157 
@Article{pmid23651157,
   Author="Davidson, E. A.  and Meyer, A. J.  and Ellefson, J. W.  and Levy, M.  and Ellington, A. D. ",
   Title="{{A}n in vitro autogene}",
   Journal="ACS Synth Biol",
   Year="2012",
   Volume="1",
   Number="5",
   Pages="190--196",
   Month="May",
   Abstract={Recent technological advances have allowed development of increasingly complex systems for in vitro evolution. Here, we describe an in vitro autogene composed of a self-amplifying T7 RNA polymerase system. Functional autogene templates in cell-free lysate produce T7 RNA polymerase, which amplifies the autogene genetic information through a positive feedback architecture. Compartmentalization of individual templates within a water-in-oil emulsion links genotype and phenotype, allowing evolution.}
}

% 23647466 
@Article{pmid23647466,
   Author="Jiang, Y. S.  and Li, B.  and Milligan, J. N.  and Bhadra, S.  and Ellington, A. D. ",
   Title="{{R}eal-time detection of isothermal amplification reactions with thermostable catalytic hairpin assembly}",
   Journal="J. Am. Chem. Soc.",
   Year="2013",
   Volume="135",
   Number="20",
   Pages="7430--7433",
   Month="May",
   Abstract={Catalytic hairpin assembly (CHA) is an enzyme-free amplification method that has previously proven useful in amplifying and transducing signals at the terminus of nucleic acid amplification reactions. Here, for the first time, we engineered CHA to be thermostable from 37 to 60 °C and in consequence have generalized its application to the real-time detection of isothermal amplification reactions. CHA circuits were designed and optimized for both high- and low-temperature rolling circle amplification (RCA) and strand displacement amplification (SDA). The resulting circuits not only increased the specificity of detection but also improved the sensitivity by as much as 25- to 10000-fold over comparable real-time detection methods. These methods have been condensed into a set of general rules for the design of thermostable CHA circuits with high signals and low noise.}
}

% 23509255 
@Article{pmid23509255,
   Author="Chen, X.  and Briggs, N.  and McLain, J. R.  and Ellington, A. D. ",
   Title="{{S}tacking nonenzymatic circuits for high signal gain}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2013",
   Volume="110",
   Number="14",
   Pages="5386--5391",
   Month="Apr",
   Abstract={Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.}
}

% 23423891 
@Article{pmid23423891,
   Author="Singh-Blom, A.  and Hughes, R. A.  and Ellington, A. D. ",
   Title="{{R}esidue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo}",
   Journal="Methods Mol. Biol.",
   Year="2013",
   Volume="978",
   Pages="93--114",
   Abstract={The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino acid substitution on protein structure and function. Herein we describe protocols to introduce unnatural amino acids into proteins using the Escherichia coli translation system either in vivo or in vitro. Special attention is paid to obtaining high levels of incorporation while maintaining high yields of protein expression.}
}

% 23405267 
@Article{pmid23405267,
   Author="Zhao, A.  and Tsechansky, M.  and Swaminathan, J.  and Cook, L.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{T}ransiently transfected purine biosynthetic enzymes form stress bodies}",
   Journal="PLoS ONE",
   Year="2013",
   Volume="8",
   Number="2",
   Pages="e56203",
   Abstract={It has been hypothesized that components of enzymatic pathways might organize into intracellular assemblies to improve their catalytic efficiency or lead to coordinate regulation. Accordingly, de novo purine biosynthesis enzymes may form a purinosome in the absence of purines, and a punctate intracellular body has been identified as the purinosome. We investigated the mechanism by which human de novo purine biosynthetic enzymes might be organized into purinosomes, especially under differing cellular conditions. Irregardless of the activity of bodies formed by endogenous enzymes, we demonstrate that intracellular bodies formed by transiently transfected, fluorescently tagged human purine biosynthesis proteins are best explained as protein aggregation.}
}

% 23382245 
@Article{pmid23382245,
   Author="Wine, Y.  and Boutz, D. R.  and Lavinder, J. J.  and Miklos, A. E.  and Hughes, R. A.  and Hoi, K. H.  and Jung, S. T.  and Horton, A. P.  and Murrin, E. M.  and Ellington, A. D.  and Marcotte, E. M.  and Georgiou, G. ",
   Title="{{M}olecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2013",
   Volume="110",
   Number="8",
   Pages="2993--2998",
   Month="Feb",
   Abstract={We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.}
}

% 23334449 
@Article{pmid23334449,
   Author="DeKosky, B. J.  and Ippolito, G. C.  and Deschner, R. P.  and Lavinder, J. J.  and Wine, Y.  and Rawlings, B. M.  and Varadarajan, N.  and Giesecke, C.  and Dorner, T.  and Andrews, S. F.  and Wilson, P. C.  and Hunicke-Smith, S. P.  and Willson, C. G.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{H}igh-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire}",
   Journal="Nat. Biotechnol.",
   Year="2013",
   Volume="31",
   Number="2",
   Pages="166--169",
   Month="Feb",
   Abstract={Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.}
}

% 23143151 
@Article{pmid23143151,
   Author="Allen, P. B.  and Chen, X.  and Ellington, A. D. ",
   Title="{{S}patial control of {D}{N}{A} reaction networks by {D}{N}{A} sequence}",
   Journal="Molecules",
   Year="2012",
   Volume="17",
   Number="11",
   Pages="13390--13402",
   Abstract={We have developed a set of DNA circuits that execute during gel electrophoresis to yield immobile, fluorescent features in the gel. The parallel execution of orthogonal circuits led to the simultaneous production of different fluorescent lines at different positions in the gel. The positions of the lines could be rationally manipulated by changing the mobilities of the reactants. The ability to program at the nanoscale so as to produce patterns at the macroscale is a step towards programmable, synthetic chemical systems for generating defined spatiotemporal patterns.}
}

% 23132138 
@Article{pmid23132138,
   Author="Jiang, Y.  and Li, B.  and Chen, X.  and Ellington, A. D. ",
   Title="{{C}oupling two different nucleic acid circuits in an enzyme-free amplifier}",
   Journal="Molecules",
   Year="2012",
   Volume="17",
   Number="11",
   Pages="13211--13220",
   Abstract={DNA circuits have proven to be useful amplifiers for diagnostic applications, in part because of their modularity and programmability. In order to determine whether different circuits could be modularly stacked, we used a catalytic hairpin assembly (CHA) circuit to initiate a hybridization chain reaction (HCR) circuit. In response to an input nucleic acid sequence, the CHA reaction accumulates immobilized duplexes and HCR elongates these duplexes. With fluorescein as a reporter each of these processes yielded 10-fold signal amplification in a convenient 96-well format. The modular circuit connections also allowed the output reporter to be readily modified to a G-quadruplex-DNAzyme that yielded a fluorescent signal.}
}

% 23104156 
@Article{pmid23104156,
   Author="Wan, Y.  and Tamuly, D.  and Allen, P. B.  and Kim, Y. T.  and Bachoo, R.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{P}roliferation and migration of tumor cells in tapered channels}",
   Journal="Biomed Microdevices",
   Year="2013",
   Volume="15",
   Number="4",
   Pages="635--643",
   Month="Aug",
   Abstract={Tumor cells depict two deviant tendencies; over-proliferation and vigorous migration. A tapered channel device is designed and fabricated for in vitro studies. We report inhibited proliferation and migration of human glioblastoma (hGBM) cells when exposed to an aptamer that is known to bind epidermal growth factor receptors (EGFR). The device is integrated with controlled ambient and microscope for providing real-time and quantitative characterization of the tumor cell behavior. The results show that hGBM cells loose proliferation and motility when exposed to the anti-EGFR aptamer. The aptamer directly inhibits and blocks EGF-induced EGFR phosphorylation. This also reduces the ability of cells to remodel their internal structure for invasion through narrow constrictions. This provides a framework for possible studies on efficacy of other inhibiting molecules.}
}

% 23057741 
@Article{pmid23057741,
   Author="O'Connell, J. D.  and Zhao, A.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{D}ynamic reorganization of metabolic enzymes into intracellular bodies}",
   Journal="Annu. Rev. Cell Dev. Biol.",
   Year="2012",
   Volume="28",
   Pages="89--111",
   Abstract={Both focused and large-scale cell biological and biochemical studies have revealed that hundreds of metabolic enzymes across diverse organisms form large intracellular bodies. These proteinaceous bodies range in form from fibers and intracellular foci--such as those formed by enzymes of nitrogen and carbon utilization and of nucleotide biosynthesis--to high-density packings inside bacterial microcompartments and eukaryotic microbodies. Although many enzymes clearly form functional mega-assemblies, it is not yet clear for many recently discovered cases whether they represent functional entities, storage bodies, or aggregates. In this article, we survey intracellular protein bodies formed by metabolic enzymes, asking when and why such bodies form and what their formation implies for the functionality--and dysfunctionality--of the enzymes that comprise them. The panoply of intracellular protein bodies also raises interesting questions regarding their evolution and maintenance within cells. We speculate on models for how such structures form in the first place and why they may be inevitable.}
}

% 23030589 
@Article{pmid23030589,
   Author="Ray, P.  and Cheek, M. A.  and Sharaf, M. L.  and Li, N.  and Ellington, A. D.  and Sullenger, B. A.  and Shaw, B. R.  and White, R. R. ",
   Title="{{A}ptamer-mediated delivery of chemotherapy to pancreatic cancer cells}",
   Journal="Nucleic Acid Ther",
   Year="2012",
   Volume="22",
   Number="5",
   Pages="295--305",
   Month="Oct",
   Abstract={Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo.}
}

% 23018995 
@Article{pmid23018995,
   Author="Opazo, F.  and Levy, M.  and Byrom, M.  and Schafer, C.  and Geisler, C.  and Groemer, T. W.  and Ellington, A. D.  and Rizzoli, S. O. ",
   Title="{{A}ptamers as potential tools for super-resolution microscopy}",
   Journal="Nat. Methods",
   Year="2012",
   Volume="9",
   Number="10",
   Pages="938--939",
   Month="Oct"
}

% 22983436 
@Article{pmid22983436,
   Author="Wan, Y.  and Liu, Y.  and Allen, P. B.  and Asghar, W.  and Mahmood, M. A.  and Tan, J.  and Duhon, H.  and Kim, Y. T.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{C}apture, isolation and release of cancer cells with aptamer-functionalized glass bead array}",
   Journal="Lab Chip",
   Year="2012",
   Volume="12",
   Number="22",
   Pages="4693--4701",
   Month="Nov",
   Abstract={Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.}
}

% 22947054 
@Article{pmid22947054,
   Author="Li, B.  and Chen, X.  and Ellington, A. D. ",
   Title="{{A}dapting enzyme-free {D}{N}{A} circuits to the detection of loop-mediated isothermal amplification reactions}",
   Journal="Anal. Chem.",
   Year="2012",
   Volume="84",
   Number="19",
   Pages="8371--8377",
   Month="Oct",
   Abstract={Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~10(9) copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a nonenzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/μL model templates (M13mp18) can produce significant signal, and both nonspecific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons.}
}

% 22938015 
@Article{pmid22938015,
   Author="Tang, H.  and Deschner, R.  and Allen, P.  and Cho, Y.  and Sermas, P.  and Maurer, A.  and Ellington, A. D.  and Willson, C. G. ",
   Title="{{A}nalysis of {D}{N}{A}-guided self-assembly of microspheres using imaging flow cytometry}",
   Journal="J. Am. Chem. Soc.",
   Year="2012",
   Volume="134",
   Number="37",
   Pages="15245--15248",
   Month="Sep",
   Abstract={Imaging flow cytometry was used to analyze the self-assembly of DNA-conjugated polystyrene microspheres. This technique enables quantitative analysis of the assembly process and thereby enables detailed analysis of the effect of structural and process variables on the assembly yield. In a demonstration of the potential of this technique, the influence of DNA strand base pair (bp) length was examined, and it was found that 50 bp was sufficient to drive the assembly of microspheres efficiently, forming not only dimers but also chainlike structures. The effect of stoichiometry on the yield was also examined. The analysis demonstrated that self-assembly of 50 bp microspheres can be driven nearly to completion by stoichiometric excess in a manner similar to Le Chatelier's principle in common chemical equilibrium.}
}

% 22927795 
@Article{pmid22927795,
   Author="Cha, H. J.  and Byrom, M.  and Mead, P. E.  and Ellington, A. D.  and Wallingford, J. B.  and Marcotte, E. M. ",
   Title="{{E}volutionarily repurposed networks reveal the well-known antifungal drug thiabendazole to be a novel vascular disrupting agent}",
   Journal="PLoS Biol.",
   Year="2012",
   Volume="10",
   Number="8",
   Pages="e1001379",
   Abstract={Studies in diverse organisms have revealed a surprising depth to the evolutionary conservation of genetic modules. For example, a systematic analysis of such conserved modules has recently shown that genes in yeast that maintain cell walls have been repurposed in vertebrates to regulate vein and artery growth. We reasoned that by analyzing this particular module, we might identify small molecules targeting the yeast pathway that also act as angiogenesis inhibitors suitable for chemotherapy. This insight led to the finding that thiabendazole, an orally available antifungal drug in clinical use for 40 years, also potently inhibits angiogenesis in animal models and in human cells. Moreover, in vivo time-lapse imaging revealed that thiabendazole reversibly disassembles newly established blood vessels, marking it as vascular disrupting agent (VDA) and thus as a potential complementary therapeutic for use in combination with current anti-angiogenic therapies. Importantly, we also show that thiabendazole slows tumor growth and decreases vascular density in preclinical fibrosarcoma xenografts. Thus, an exploration of the evolutionary repurposing of gene networks has led directly to the identification of a potential new therapeutic application for an inexpensive drug that is already approved for clinical use in humans.}
}

% 22894754 
@Article{pmid22894754,
   Author="Li, B.  and Jiang, Y.  and Chen, X.  and Ellington, A. D. ",
   Title="{{P}robing spatial organization of {D}{N}{A} strands using enzyme-free hairpin assembly circuits}",
   Journal="J. Am. Chem. Soc.",
   Year="2012",
   Volume="134",
   Number="34",
   Pages="13918--13921",
   Month="Aug",
   Abstract={Catalyzed hairpin assembly (CHA) is a robust enzyme-free signal-amplification reaction that has a wide range of potential applications, especially in biosensing. Although most studies of the analytical applications of CHA have focused on the measurement of concentrations of biomolecules, we show here that CHA can also be used to probe the spatial organization of biomolecules such as single-stranded DNA. The basis of such detection is the fact that a DNA structure that brings a toehold and a branch-migration domain into close proximity can catalyze the CHA reaction. We quantitatively studied this phenomenon and applied it to the detection of domain reorganization that occurs during DNA self-assembly processes such as the hybridization chain reaction (HCR). We also show that CHA circuits can be designed to detect certain types of hybridization defects. This principle allowed us to develop a "signal on" assay that can simultaneously respond to multiple types of mutations in a DNA strand in one simple reaction, which is of great interest in genotyping and molecular diagnostics. These findings highlight the potential impacts of DNA circuitry on DNA nanotechnology and provide new tools for further development of these fields.}
}

% 22891822 
@Article{pmid22891822,
   Author="Meyer, A. J.  and Ellefson, J. W.  and Ellington, A. D. ",
   Title="{{A}biotic self-replication}",
   Journal="Acc. Chem. Res.",
   Year="2012",
   Volume="45",
   Number="12",
   Pages="2097--2105",
   Month="Dec",
   Abstract={The key to the origins of life is the replication of information. Linear polymers such as nucleic acids that both carry information and can be replicated are currently what we consider to be the basis of living systems. However, these two properties are not necessarily coupled. The ability to mutate in a discrete or quantized way, without frequent reversion, may be an additional requirement for Darwinian evolution, in which case the notion that Darwinian evolution defines life may be less of a tautology than previously thought. In this Account, we examine a variety of in vitro systems of increasing complexity, from simple chemical replicators up to complex systems based on in vitro transcription and translation. Comparing and contrasting these systems provides an interesting window onto the molecular origins of life. For nucleic acids, the story likely begins with simple chemical replication, perhaps of the form A + B → T, in which T serves as a template for the joining of A and B. Molecular variants capable of faster replication would come to dominate a population, and the development of cycles in which templates could foster one another's replication would have led to increasingly complex replicators and from thence to the initial genomes. The initial genomes may have been propagated by RNA replicases, ribozymes capable of joining oligonucleotides and eventually polymerizing mononucleotide substrates. As ribozymes were added to the genome to fill gaps in the chemistry necessary for replication, the backbone of a putative RNA world would have emerged. It is likely that such replicators would have been plagued by molecular parasites, which would have been passively replicated by the RNA world machinery without contributing to it. These molecular parasites would have been a major driver for the development of compartmentalization/cellularization, as more robust compartments could have outcompeted parasite-ridden compartments. The eventual outsourcing of metabolic functions (including the replication of nucleic acids) to more competent protein enzymes would complete the journey from an abiotic world to the molecular biology we see today.}
}

% 22870859 
@Article{pmid22870859,
   Author="Hughes, R. A.  and Miklos, A. E.  and Ellington, A. D. ",
   Title="{{E}nrichment of error-free synthetic {D}{N}{A} sequences by {C}{E}{L} {I} nuclease}",
   Journal="Curr Protoc Mol Biol",
   Year="2012",
   Volume="Chapter 3",
   Pages="Unit3.24",
   Month="Jul",
   Abstract={As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment.}
}

% 22870858 
@Article{pmid22870858,
   Author="Miklos, A. E.  and Hughes, R. A.  and Ellington, A. D. ",
   Title="{{D}esign and assembly of large synthetic {D}{N}{A} constructs}",
   Journal="Curr Protoc Mol Biol",
   Year="2012",
   Volume="Chapter 3",
   Pages="Unit3.23",
   Month="Jul",
   Abstract={The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides.}
}

% 22737599 
@Article{pmid22737599,
   Author="Romero, P. A.  and Stone, E.  and Lamb, C.  and Chantranupong, L.  and Krause, A.  and Miklos, A. E.  and Hughes, R. A.  and Fechtel, B.  and Ellington, A. D.  and Arnold, F. H.  and Georgiou, G. ",
   Title="{{S}{C}{H}{E}{M}{A}-designed variants of human {A}rginase {I} and {I}{I} reveal sequence elements important to stability and catalysis}",
   Journal="ACS Synth Biol",
   Year="2012",
   Volume="1",
   Number="6",
   Pages="221--228",
   Month="Jun",
   Abstract={Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.}
}

% 22729075 
@Article{pmid22729075,
   Author="Allen, P. B.  and Arshad, S. A.  and Li, B.  and Chen, X.  and Ellington, A. D. ",
   Title="{{D}{N}{A} circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform}",
   Journal="Lab Chip",
   Year="2012",
   Volume="12",
   Number="16",
   Pages="2951--2958",
   Month="Aug",
   Abstract={This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.}
}

% 22706642 
@Article{pmid22706642,
   Author="Ilyas, A.  and Asghar, W.  and Allen, P. B.  and Duhon, H.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{E}lectrical detection of cancer biomarker using aptamers with nanogap break-junctions}",
   Journal="Nanotechnology",
   Year="2012",
   Volume="23",
   Number="27",
   Pages="275502",
   Month="Jul",
   Abstract={Epidermal growth factor receptor (EGFR) is a cell surface protein overexpressed in cancerous cells. It is known to be the most common oncogene. EGFR concentration also increases in the serum of cancer patients. The detection of small changes in the concentration of EGFR can be critical for early diagnosis, resulting in better treatment and improved survival rate of cancer patients. This article reports an RNA aptamer based approach to selectively capture EGFR protein and an electrical scheme for its detection. Pairs of gold electrodes with nanometer separation were made through confluence of focused ion beam scratching and electromigration. The aptamer was hybridized to a single stranded DNA molecule, which in turn was immobilized on the SiO(2) surface between the gold nanoelectrodes. The selectivity of the aptamer was demonstrated by using control chips with mutated non-selective aptamer and with no aptamer. Surface functionalization was characterized by optical detection and two orders of magnitude increase in direct current (DC) was measured when selective capture of EGFR occurred. This represents an electronic biosensor for the detection of proteins of interest for medical applications.}
}

% 22670809 
@Article{pmid22670809,
   Author="Zhang, M.  and Wang, X. J.  and Chen, X.  and Bowman, M. E.  and Luo, Y.  and Noel, J. P.  and Ellington, A. D.  and Etzkorn, F. A.  and Zhang, Y. ",
   Title="{{S}tructural and kinetic analysis of prolyl-isomerization/phosphorylation cross-talk in the {C}{T}{D} code}",
   Journal="ACS Chem. Biol.",
   Year="2012",
   Volume="7",
   Number="8",
   Pages="1462--1470",
   Month="Aug",
   Abstract={The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). CTD regulation of transcription is mediated by both phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, and thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.}
}

% 22558161 
@Article{pmid22558161,
   Author="Ippolito, G. C.  and Hoi, K. H.  and Reddy, S. T.  and Carroll, S. M.  and Ge, X.  and Rogosch, T.  and Zemlin, M.  and Shultz, L. D.  and Ellington, A. D.  and Vandenberg, C. L.  and Georgiou, G. ",
   Title="{{A}ntibody repertoires in humanized {N}{O}{D}-scid-{I}{L}2{R}γ(null) mice and human {B} cells reveals human-like diversification and tolerance checkpoints in the mouse}",
   Journal="PLoS ONE",
   Year="2012",
   Volume="7",
   Number="4",
   Pages="e35497",
   Abstract={Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγ(null) engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγ(null) mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential D(H)-J(H) pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.}
}

% 22520751 
@Article{pmid22520751,
   Author="Miklos, A. E.  and Kluwe, C.  and Der, B. S.  and Pai, S.  and Sircar, A.  and Hughes, R. A.  and Berrondo, M.  and Xu, J.  and Codrea, V.  and Buckley, P. E.  and Calm, A. M.  and Welsh, H. S.  and Warner, C. R.  and Zacharko, M. A.  and Carney, J. P.  and Gray, J. J.  and Georgiou, G.  and Kuhlman, B.  and Ellington, A. D. ",
   Title="{{S}tructure-based design of supercharged, highly thermoresistant antibodies}",
   Journal="Chem. Biol.",
   Year="2012",
   Volume="19",
   Number="4",
   Pages="449--455",
   Month="Apr",
   Abstract={Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ≈30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.}
}

% 22491026 
@Article{pmid22491026,
   Author="Lu, W. C.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of proteins via emulsion compartments}",
   Journal="Methods",
   Year="2013",
   Volume="60",
   Number="1",
   Pages="75--80",
   Month="Mar",
   Abstract={In vitro compartmentalization (IVC) is a method to generate numerous, small, aqueous compartments (up to 10(10) compartments per ml) by mixing water, surfactants, and oil. The water phase is surrounded by surfactants and an oil phase, and to a first approximation each water-in-oil compartment is like an artificial cell. By introducing single genes into compartments that are competent for transcription and translation, these cell-like compartments can synthesize RNA protein variants in libraries. Screening or selecting for function has in turn led to schemes for the directed evolution of biomolecules. However, IVC selections can cover larger library sizes, and provide greater control over selection conditions and stringencies. The key issue in designing and executing IVC selections is how to couple genotype and phenotype, and in this review we have organized and presented a variety of mechanisms by which proteins and RNA can attach to or amplify their own templates following emulsification and selection.}
}

% 22233578 
@Article{pmid22233578,
   Author="Magalhaes, M. L.  and Byrom, M.  and Yan, A.  and Kelly, L.  and Li, N.  and Furtado, R.  and Palliser, D.  and Ellington, A. D.  and Levy, M. ",
   Title="{{A} general {R}{N}{A} motif for cellular transfection}",
   Journal="Mol. Ther.",
   Year="2012",
   Volume="20",
   Number="3",
   Pages="616--624",
   Month="Mar",
   Abstract={We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.}
}

% 22223052 
@Article{pmid22223052,
   Author="Cho, E. J.  and Xia, S.  and Ma, L. C.  and Robertus, J.  and Krug, R. M.  and Anslyn, E. V.  and Montelione, G. T.  and Ellington, A. D. ",
   Title="{{I}dentification of influenza virus inhibitors targeting {N}{S}1{A} utilizing fluorescence polarization-based high-throughput assay}",
   Journal="J Biomol Screen",
   Year="2012",
   Volume="17",
   Number="4",
   Pages="448--459",
   Month="Apr",
   Abstract={This article describes the development of a simple and robust fluorescence polarization (FP)-based binding assay and adaptation to high-throughput identification of small molecules blocking dsRNA binding to NS1A protein (nonstructural protein 1 from type A influenza strains). This homogeneous assay employs fluorescein-labeled 16-mer dsRNA and full-length NS1A protein tagged with glutathione S-transferase to monitor the changes in FP and fluorescence intensity simultaneously. The assay was optimized for high-throughput screening in a 384-well format and achieved a z' score greater than 0.7. Its feasibility for high-throughput screening was demonstrated using the National Institutes of Health clinical collection. Six of 446 small molecules were identified as possible ligands in an initial screening. A series of validation tests confirmed epigallocatechine gallate (EGCG) to be active in the submicromolar range. A mechanism of EGCG inhibition involving interaction with the dsRNA-binding motif of NS1A, including Arg38, was proposed. This structural information is anticipated to provide a useful basis for the modeling of antiflu therapeutic reagents. Overall, the FP-based binding assay demonstrated its superior capability for simple, rapid, inexpensive, and robust identification of NS1A inhibitors and validation of their activity targeting NS1A.}
}

% 22019591 
@Article{pmid22019591,
   Author="Alicea, I.  and Marvin, J. S.  and Miklos, A. E.  and Ellington, A. D.  and Looger, L. L.  and Schreiter, E. R. ",
   Title="{{S}tructure of the {E}scherichia coli phosphonate binding protein {P}hn{D} and rationally optimized phosphonate biosensors}",
   Journal="J. Mol. Biol.",
   Year="2011",
   Volume="414",
   Number="3",
   Pages="356--369",
   Month="Dec",
   Abstract={The phnD gene of Escherichia coli encodes the periplasmic binding protein of the phosphonate (Pn) uptake and utilization pathway. We have crystallized and determined structures of E. coli PhnD (EcPhnD) in the absence of ligand and in complex with the environmentally abundant 2-aminoethylphosphonate (2AEP). Similar to other bacterial periplasmic binding proteins, 2AEP binds near the center of mass of EcPhnD in a cleft formed between two lobes. Comparison of the open, unliganded structure with the closed 2AEP-bound structure shows that the two lobes pivot around a hinge by ~70° between the two states. Extensive hydrogen bonding and electrostatic interactions stabilize 2AEP, which binds to EcPhnD with low nanomolar affinity. These structures provide insight into Pn uptake by bacteria and facilitated the rational design of high signal-to-noise Pn biosensors based on both coupled small-molecule dyes and autocatalytic fluorescent proteins.}
}

% 21938060 
@Article{pmid21938060,
   Author="Enyeart, P. J.  and Ellington, A. D. ",
   Title="{{S}ynthetic biology: a yeast for all reasons}",
   Journal="Nature",
   Year="2011",
   Volume="477",
   Number="7365",
   Pages="413--414",
   Month="Sep"
}

% 21766299 
@Article{pmid21766299,
   Author="Wan, Y.  and Mahmood, M. A.  and Li, N.  and Allen, P. B.  and Kim, Y. T.  and Bachoo, R.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{N}anotextured substrates with immobilized aptamers for cancer cell isolation and cytology}",
   Journal="Cancer",
   Year="2012",
   Volume="118",
   Number="4",
   Pages="1145--1154",
   Month="Feb",
   Abstract={The detection of a small number of circulating tumor cells (CTCs) is important, especially in the early stages of cancer. Small numbers of CTCs are hard to detect, because very few approaches are sensitive enough to differentiate these from the pool of other cells. Improving the affinity of a selective, surface-functionalized molecule is important given the scarcity of CTCs in vivo. There are several proteins and aptamers that provide such high affinity; however, using surface nanotexturing increases this affinity even further.\\ The authors report an approach to improve the affinity of tumor cell capture by using novel aptamers against cell membrane overexpressed epidermal growth factor receptors (EGFRs) on a nanotextured polydimethylsiloxane (PDMS) substrate. Surface-immobilized aptamers were used to specifically capture tumor cells from physiologic samples.\\ The nanotexturing of PDMS increased surface roughness at the nanoscale. This increased the effective surface area and resulted in a significantly higher degree of surface functionalization. The phenomenon resulted in increased density of immobilized EGFR-specific RNA aptamer molecules and provided significantly higher efficiency to capture cancer cells from a mixture. The data indicated that CTCs could be captured and enriched, leading to higher yield yet higher background.\\ A comparison between glass slides, plain PDMS, and nanotextured PDMS functionalized with aptamers demonstrated that a 2-fold approach of using aptamers on nanotextured PDMS can be important for cancer cytology devices, and especially for the idea of a "lab-on-chip," toward higher yield in capture efficiency.}
}

% 21693555 
@Article{pmid21693555,
   Author="Li, B.  and Ellington, A. D.  and Chen, X. ",
   Title="{{R}ational, modular adaptation of enzyme-free {D}{N}{A} circuits to multiple detection methods}",
   Journal="Nucleic Acids Res.",
   Year="2011",
   Volume="39",
   Number="16",
   Pages="e110",
   Month="Sep",
   Abstract={Signal amplification is a key component of molecular detection. Enzyme-free signal amplification is especially appealing for the development of low-cost, point-of-care diagnostics. It has been previously shown that enzyme-free DNA circuits with signal-amplification capacity can be designed using a mechanism called 'catalyzed hairpin assembly'. However, it is unclear whether the efficiency and modularity of such circuits is suitable for multiple analytical applications. We have therefore designed and characterized a simplified DNA circuit based on catalyzed hairpin assembly, and applied it to multiple different analytical formats, including fluorescent, colorimetric, and electrochemical and signaling. By optimizing the design of previous hairpin-based catalytic assemblies we found that our circuit has almost zero background and a high catalytic efficiency, with a k(cat) value above 1 min(-1). The inherent modularity of the circuit allowed us to readily adapt our circuit to detect both RNA and small molecule analytes. Overall, these data demonstrate that catalyzed hairpin assembly is suitable for analyte detection and signal amplification in a 'plug-and-play' fashion.}
}

% 21687663 
@Article{pmid21687663,
   Author="Li, N.  and Nguyen, H. H.  and Byrom, M.  and Ellington, A. D. ",
   Title="{{I}nhibition of cell proliferation by an anti-{E}{G}{F}{R} aptamer}",
   Journal="PLoS ONE",
   Year="2011",
   Volume="6",
   Number="6",
   Pages="e20299",
   Abstract={Aptamers continue to receive interest as potential therapeutic agents for the treatment of diseases, including cancer. In order to determine whether aptamers might eventually prove to be as useful as other clinical biopolymers, such as antibodies, we selected aptamers against an important clinical target, human epidermal growth factor receptor (hEGFR). The initial selection yielded only a single clone that could bind to hEGFR, but further mutation and optimization yielded a family of tight-binding aptamers. One of the selected aptamers, E07, bound tightly to the wild-type receptor (K(d) = 2.4 nM). This aptamer can compete with EGF for binding, binds to a novel epitope on EGFR, and also binds a deletion mutant, EGFRvIII, that is commonly found in breast and lung cancers, and especially in grade IV glioblastoma multiforme, a cancer which has for the most part proved unresponsive to current therapies. The aptamer binds to cells expressing EGFR, blocks receptor autophosphorylation, and prevents proliferation of tumor cells in three-dimensional matrices. In short, the aptamer is a promising candidate for further development as an anti-tumor therapeutic. In addition, Aptamer E07 is readily internalized into EGFR-expressing cells, raising the possibility that it might be used to escort other anti-tumor or contrast agents.}
}

% 21654670 
@Article{pmid21654670,
   Author="Meyer, A. J.  and Ellington, A. D. ",
   Title="{{M}olecular evolution picks up the {P}{A}{C}{E}}",
   Journal="Nat. Biotechnol.",
   Year="2011",
   Volume="29",
   Number="6",
   Pages="502--503",
   Month="Jun"
}

% 21601682 
@Article{pmid21601682,
   Author="Hughes, R. A.  and Miklos, A. E.  and Ellington, A. D. ",
   Title="{{G}ene synthesis: methods and applications}",
   Journal="Meth. Enzymol.",
   Year="2011",
   Volume="498",
   Pages="277--309",
   Abstract={DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.}
}

% 21338059 
@Article{pmid21338059,
   Author="Allen, P. B.  and Ellington, A. D. ",
   Title="{{S}equential injection analysis for optimization of molecular biology reactions}",
   Journal="Anal. Chem.",
   Year="2011",
   Volume="83",
   Number="6",
   Pages="2194--2200",
   Month="Mar",
   Abstract={In order to automate the optimization of complex biochemical and molecular biology reactions, we developed a sequential injection analysis (SIA) device and combined this with a design of experiment (DOE) algorithm. This combination of hardware and software automatically explores the parameter space of the reaction and provides continuous feedback for optimizing reaction conditions. As an example, we optimized the endonuclease digest of a fluorogenic substrate and showed that the optimized reaction conditions also applied to the digest of the substrate outside of the device and to the digest of a plasmid. The sequential technique quickly arrived at optimized reaction conditions with less reagent use than a batch process (such as a fluid handling robot exploring multiple reaction conditions in parallel) would have. The device and method should now be amenable to much more complex molecular biology reactions whose variable spaces are correspondingly larger.}
}

% 21299200 
@Article{pmid21299200,
   Author="Hargrove, A. E.  and Ellington, A. D.  and Anslyn, E. V.  and Sessler, J. L. ",
   Title="{{C}hemical functionalization of oligodeoxynucleotides with multiple boronic acids for the polyvalent binding of saccharides}",
   Journal="Bioconjug. Chem.",
   Year="2011",
   Volume="22",
   Number="3",
   Pages="388--396",
   Month="Mar",
   Abstract={A novel saccharide host containing four boronic acid recognition units on a single DNA duplex terminus was constructed. This construct allowed boronic acid sugar recognition in the context of double-stranded DNA to be established while highlighting the benefits of multivalency. Following the solid-phase synthesis of a bis-boronic acid tag, two end-functionalized oligonucleotides with complementary sequences were functionalized through amide ligation. By annealing the boronic acid-DNA conjugates, a tetra-boronic acid DNA duplex was assembled. The saccharide binding ability of this tetra-boronic acid host was revealed through cellulose paper chromatography in the absence and presence of various saccharides. While no appreciable saccharide binding was seen in the case of a bis-boronic DNA conjugate, the increased migration of the tetra-boronic acid host relative to the control sequences in the presence of selected monosaccharides underscored the importance of multivalent effects. We thus identified a requirement for multiple recognition sites in these conjugate systems and expect the results to facilitate future efforts toward applying synthetic recognition systems to the realm of macromolecules.}
}

% 23850755 
@Article{pmid23850755,
   Author="Manickam, A.  and Chevalier, A.  and McDermott, M.  and Ellington, A. D.  and Hassibi, A. ",
   Title="{{A} {C}{M}{O}{S} {E}lectrochemical {I}mpedance {S}pectroscopy ({E}{I}{S}) {B}iosensor {A}rray}",
   Journal="IEEE Trans Biomed Circuits Syst",
   Year="2010",
   Volume="4",
   Number="6",
   Pages="379--390",
   Month="Dec",
   Abstract={In this paper, we present a fully integrated biosensor 10 × 10 array in a standard complementary metal-oxide semiconducor process, which takes advantage of electrochemical impedance spectroscopy (EIS). We also show that this system is able to detect various biological analytes, such as DNA and proteins, in real time and without the need for molecular labels. In each pixel of this array, we implement a biocompatible Au electrode transducer and embedded sensor circuitry which takes advantage of the coherent detector to measure the impedance of the associated electrode-electrolyte interface. This chip is capable of concurrently measuring admittance values as small as 10(-8) Ω(-1) within the array with the detection dynamic range of more than 90 dB in the frequency range of 10 Hz-50 MHz.}
}

% 21062984 
@Article{pmid21062984,
   Author="Wan, Y.  and Kim, Y. T.  and Li, N.  and Cho, S. K.  and Bachoo, R.  and Ellington, A. D.  and Iqbal, S. M. ",
   Title="{{S}urface-immobilized aptamers for cancer cell isolation and microscopic cytology}",
   Journal="Cancer Res.",
   Year="2010",
   Volume="70",
   Number="22",
   Pages="9371--9380",
   Month="Nov",
   Abstract={Exposing rare but highly malignant tumor cells that migrate from the primary tumor mass into adjacent tissue(s) or circulate in the bloodstream is critical for early detection and effective intervention(s). Here, we report on an aptamer-based strategy directed against epidermal growth factor receptor (EGFR), the most common oncogene in glioblastoma (GBM), to detect these deadly tumor cells. GBMs are characterized by diffuse infiltration into normal brain regions, and the inability to detect GBM cells renders the disease surgically incurable with a median survival of just 14.2 months. To test the sensitivity and specificity of our platform, anti-EGFR RNA aptamers were immobilized on chemically modified glass surfaces. Cells tested included primary human GBM cells expressing high levels of the wild-type EGFR, as well as genetically engineered murine glioma cells overexpressing the most common EGFR mutant (EGFRvIII lacking exons 2-7) in Ink4a/Arf-deficient astrocytes. We found that surfaces functionalized with anti-EGFR aptamers could capture both the human and murine GBM cells with high sensitivity and specificity. Our findings show how novel aptamer substrates could be used to determine whether surgical resection margins are free of tumor cells, or more widely for detecting tumor cells circulating in peripheral blood to improve early detection and/or monitoring residual disease after treatment.}
}

% 21060734 
@Article{pmid21060734,
   Author="Connell, J. L.  and Wessel, A. K.  and Parsek, M. R.  and Ellington, A. D.  and Whiteley, M.  and Shear, J. B. ",
   Title="{{P}robing prokaryotic social behaviors with bacterial "lobster traps"}",
   Journal="MBio",
   Year="2010",
   Volume="1",
   Number="4",
   Pages=" ",
   Abstract={Bacteria are social organisms that display distinct behaviors/phenotypes when present in groups. These behaviors include the abilities to construct antibiotic-resistant sessile biofilm communities and to communicate with small signaling molecules (quorum sensing [QS]). Our understanding of biofilms and QS arises primarily from in vitro studies of bacterial communities containing large numbers of cells, often greater than 10(8) bacteria; however, in nature, bacteria often reside in dense clusters (aggregates) consisting of significantly fewer cells. Indeed, bacterial clusters containing 10(1) to 10(5) cells are important for transmission of many bacterial pathogens. Here, we describe a versatile strategy for conducting mechanistic studies to interrogate the molecular processes controlling antibiotic resistance and QS-mediated virulence factor production in high-density bacterial clusters. This strategy involves enclosing a single bacterium within three-dimensional picoliter-scale microcavities (referred to as bacterial "lobster traps") defined by walls that are permeable to nutrients, waste products, and other bioactive small molecules. Within these traps, bacteria divide normally into extremely dense (10(12) cells/ml) clonal populations with final population sizes similar to that observed in naturally occurring bacterial clusters. Using these traps, we provide strong evidence that within low-cell-number/high-density bacterial clusters, QS is modulated not only by bacterial density but also by population size and flow rate of the surrounding medium. We also demonstrate that antibiotic resistance develops as cell density increases, with as few as ~150 confined bacteria exhibiting an antibiotic-resistant phenotype similar to biofilm bacteria. Together, these findings provide key insights into clinically relevant phenotypes in low-cell-number/high-density bacterial populations.}
}

% 20947753 
@Article{pmid20947753,
   Author="Kluwe, C.  and Ellington, A. D. ",
   Title="{{E}volution. {R}{N}{A} {G}{P}{S}}",
   Journal="Science",
   Year="2010",
   Volume="330",
   Number="6002",
   Pages="330--331",
   Month="Oct"
}

% 20802495 
@Article{pmid20802495,
   Author="Reddy, S. T.  and Ge, X.  and Miklos, A. E.  and Hughes, R. A.  and Kang, S. H.  and Hoi, K. H.  and Chrysostomou, C.  and Hunicke-Smith, S. P.  and Iverson, B. L.  and Tucker, P. W.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{M}onoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells}",
   Journal="Nat. Biotechnol.",
   Year="2010",
   Volume="28",
   Number="9",
   Pages="965--969",
   Month="Sep",
   Abstract={Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.}
}

% 20636061 
@Article{pmid20636061,
   Author="Cho, E. J.  and Lee, J. W.  and Ellington, A. D. ",
   Title="{{A}pplications of aptamers as sensors}",
   Journal="Annu Rev Anal Chem (Palo Alto Calif)",
   Year="2009",
   Volume="2",
   Pages="241--264",
   Abstract={Aptamers are ligand-binding nucleic acids whose affinities and selectivities can rival those of antibodies. They have been adapted to analytical applications not only as alternatives to antibodies, but as unique reagents in their own right. In particular, aptamers can be readily site-specifically modified during chemical or enzymatic synthesis to incorporate particular reporters, linkers, or other moieties. Also, aptamer secondary structures can be engineered to undergo analyte-dependent conformational changes, which, in concert with the ability to specifically place chemical agents, opens up a wealth of possible signal transduction schemas, irrespective of whether the detection modality is optical, electrochemical, or mass based. Finally, because aptamers are nucleic acids, they are readily adapted to sequence- (and hence signal-) amplification methods. However, application of aptamers without a basic knowledge of their biochemistry or technical requirements can cause serious analytical difficulties.}
}

% 20571084 
@Article{pmid20571084,
   Author="Hughes, R. A.  and Ellington, A. D. ",
   Title="{{R}ational design of an orthogonal tryptophanyl nonsense suppressor t{R}{N}{A}}",
   Journal="Nucleic Acids Res.",
   Year="2010",
   Volume="38",
   Number="19",
   Pages="6813--6830",
   Month="Oct",
   Abstract={While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.}
}

% 20538451 
@Article{pmid20538451,
   Author="Chen, X.  and Ellington, A. D. ",
   Title="{{S}haping up nucleic acid computation}",
   Journal="Curr. Opin. Biotechnol.",
   Year="2010",
   Volume="21",
   Number="4",
   Pages="392--400",
   Month="Aug",
   Abstract={Nucleic acid-based nanotechnology has always been perceived as novel, but has begun to move from theoretical demonstrations to practical applications. In particular, the large address spaces available to nucleic acids can be exploited to encode algorithms and/or act as circuits and thereby process molecular information. In this review we not only revisit several milestones in the field of nucleic acid-based computation, but also highlight how the prospects for nucleic acid computation go beyond just a large address space. Functional nucleic acid elements (aptamers, ribozymes, and deoxyribozymes) can serve as inputs and outputs to the environment, and can act as logical elements. Into the future, the chemical dynamics of nucleic acids may prove as useful as hybridization for computation.}
}

% 20501662 
@Article{pmid20501662,
   Author="Tipps, M. E.  and Lawshe, J. E.  and Ellington, A. D.  and Mihic, S. J. ",
   Title="{{I}dentification of novel specific allosteric modulators of the glycine receptor using phage display}",
   Journal="J. Biol. Chem.",
   Year="2010",
   Volume="285",
   Number="30",
   Pages="22840--22845",
   Month="Jul",
   Abstract={The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related gamma-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.}
}

% 20300533 
@Article{pmid20300533,
   Author="Wang, Y.  and Khaing, Z. Z.  and Li, N.  and Hall, B.  and Schmidt, C. E.  and Ellington, A. D. ",
   Title="{{A}ptamer antagonists of myelin-derived inhibitors promote axon growth}",
   Journal="PLoS ONE",
   Year="2010",
   Volume="5",
   Number="3",
   Pages="e9726",
   Abstract={Myelin of the adult central nervous system (CNS) is one of the major sources of inhibitors of axon regeneration following injury. The three known myelin-derived inhibitors (Nogo, MAG, and OMgp) bind with high affinity to the Nogo-66 receptor (NgR) on axons and limit neurite outgrowth. Here we show that RNA aptamers can be generated that bind with high affinity to NgR, compete with myelin-derived inhibitors for binding to NgR, and promote axon elongation of neurons in vitro even in the presence of these inhibitors. Aptamers may have key advantages over protein antagonists, including low immunogenicity and the possibility of ready modification during chemical synthesis for stability, signaling, or immobilization. This first demonstration that aptamers can directly influence neuronal function suggests that aptamers may prove useful for not only healing spinal cord and other neuronal damage, but may be more generally useful as neuromodulators.}
}

% 20201028 
@Article{pmid20201028,
   Author="Hall, B.  and Arshad, S.  and Seo, K.  and Bowman, C.  and Corley, M.  and Jhaveri, S. D.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2010",
   Volume="Chapter 9",
   Pages="1--27",
   Month="Mar",
   Abstract={This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.}
}

% 20198660 
@Article{pmid20198660,
   Author="Ge, X.  and Mazor, Y.  and Hunicke-Smith, S. P.  and Ellington, A. D.  and Georgiou, G. ",
   Title="{{R}apid construction and characterization of synthetic antibody libraries without {D}{N}{A} amplification}",
   Journal="Biotechnol. Bioeng.",
   Year="2010",
   Volume="106",
   Number="3",
   Pages="347--357",
   Month="Jun",
   Abstract={We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.}
}

% 20129797 
@Article{pmid20129797,
   Author="Gardner, M. W.  and Li, N.  and Ellington, A. D.  and Brodbelt, J. S. ",
   Title="{{I}nfrared multiphoton dissociation of small-interfering {R}{N}{A} anions and cations}",
   Journal="J. Am. Soc. Mass Spectrom.",
   Year="2010",
   Volume="21",
   Number="4",
   Pages="580--591",
   Month="Apr",
   Abstract={Infrared multiphoton dissociation (IRMPD) on a linear ion trap mass spectrometer is applied for the sequencing of small interfering RNA (siRNA). Both single-strand siRNAs and duplex siRNA were characterized by IRMPD, and the results were compared with that obtained by traditional ion trap-based collision induced dissociation (CID). The single-strand siRNA anions were observed to dissociate via cleavage of the 5' P-O bonds yielding c- and y-type product ions as well as undergo neutral base loss. Full sequence coverage of the siRNA anions was obtained by both IRMPD and CID. While the CID mass spectra were dominated by base loss ions, accounting for approximately 25% to 40% of the product ion current, these ions were eliminated through secondary dissociation by increasing the irradiation time in the IRMPD mass spectra to produce higher abundances of informative sequence ions. With longer irradiation times, however, internal ions corresponding to cleavage of two 5' P-O bonds began to populate the product ion mass spectra as well as higher abundances of [a - Base] and w-type ions. IRMPD of siRNA cations predominantly produced c- and y-type ions with minimal contributions of [a - Base] and w-type ions to the product ion current; the presence of only two complementary series of product ions in the IRMPD mass spectra simplified spectral interpretation. In addition, IRMPD produced high abundances of protonated nucleobases, [G + H](+), [A + H](+), and [C + H](+), which were not detected in the CID mass spectra due to the low-mass cut-off associated with conventional CID in ion traps. CID and IRMPD using short irradiation times of duplex siRNA resulted in strand separation, similar to the dissociation trends observed for duplex DNA. With longer irradiation times, however, the individual single-strands underwent secondary dissociation to yield informative sequence ions not obtained by CID.}
}

% 20066302 
@Article{pmid20066302,
   Author="Li, N.  and Larson, T.  and Nguyen, H. H.  and Sokolov, K. V.  and Ellington, A. D. ",
   Title="{{D}irected evolution of gold nanoparticle delivery to cells}",
   Journal="Chem. Commun. (Camb.)",
   Year="2010",
   Volume="46",
   Number="3",
   Pages="392--394",
   Month="Jan",
   Abstract={A newly selected anti-receptor (anti-EGFR) aptamer was conjugated to gold nanoparticles via a facile hybridization method and was found to specifically and quantitatively direct the delivery of gold nanoparticles to cells expressing EGFR through receptor-mediated endocytosis.}
}

% 20041206 
@Article{pmid20041206,
   Author="Chen, X.  and Ellington, A. D. ",
   Title="{{D}esign principles for ligand-sensing, conformation-switching ribozymes}",
   Journal="PLoS Comput. Biol.",
   Year="2009",
   Volume="5",
   Number="12",
   Pages="e1000620",
   Month="Dec",
   Abstract={Nucleic acid sensor elements are proving increasingly useful in biotechnology and biomedical applications. A number of ligand-sensing, conformational-switching ribozymes (also known as allosteric ribozymes or aptazymes) have been generated by some combination of directed evolution or rational design. Such sensor elements typically fuse a molecular recognition domain (aptamer) with a catalytic signal generator (ribozyme). Although the rational design of aptazymes has begun to be explored, the relationships between the thermodynamics of aptazyme conformational changes and aptazyme performance in vitro and in vivo have not been examined in a quantitative framework. We have therefore developed a quantitative and predictive model for aptazymes as biosensors in vitro and as riboswitches in vivo. In the process, we have identified key relationships (or dimensionless parameters) that dictate aptazyme performance, and in consequence, established equations for precisely engineering aptazyme function. In particular, our analysis quantifies the intrinsic trade-off between ligand sensitivity and the dynamic range of activity. We were also able to determine how in vivo parameters, such as mRNA degradation rates, impact the design and function of aptazymes when used as riboswitches. Using this theoretical framework we were able to achieve quantitative agreement between our models and published data. In consequence, we are able to suggest experimental guidelines for quantitatively predicting the performance of aptazyme-based riboswitches. By identifying factors that limit the performance of previously published systems we were able to generate immediately testable hypotheses for their improvement. The robust theoretical framework and identified optimization parameters should now enable the precision design of aptazymes for biotechnological and clinical applications.}
}

% 20013786 
@Article{pmid20013786,
   Author="Hall, B.  and Micheletti, J. M.  and Satya, P.  and Ogle, K.  and Pollard, J.  and Ellington, A. D. ",
   Title="{{D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2009",
   Volume="Chapter 9",
   Pages="Unit 9.2",
   Month="Dec",
   Abstract={Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.}
}

% 19947589 
@Article{pmid19947589,
   Author="Lee, J.  and Icoz, K.  and Roberts, A.  and Ellington, A. D.  and Savran, C. A. ",
   Title="{{D}iffractometric detection of proteins using microbead-based rolling circle amplification}",
   Journal="Anal. Chem.",
   Year="2010",
   Volume="82",
   Number="1",
   Pages="197--202",
   Month="Jan",
   Abstract={We present a robust, sensitive, fluorescent- or radiolabel-free self-assembled optical diffraction biosensor that utilizes rolling circle amplification (RCA) and magnetic microbeads as a signal enhancement method. An aptamer-based sandwich assay was performed on microcontact-printed streptavidin arranged in 15 microm wide alternating lines and could specifically capture and detect platelet-derived growth factor B-chain (PDGF-BB). An aptamer served as a template for the ligation of a padlock probe, and the circularized probe could in turn be used as a template for RCA. The concatameric RCA product hybridized to biotinylated oligonuclotides which then captured streptavidin-labeled magnetic beads. In consequence, the signal from the captured PDGF-BB was amplified via the concatameric RCA product, and the diffraction gratings on the printed areas produced varying intensities of diffraction modes. The detected diffraction intensity and the density of the microbeads on the surface varied as a function of PDGF-BB concentration. Our results demonstrate a robust biosensing platform that is easy to construct and use and devoid of fluorescence microscopy. The self-assembled bead patterns allow both a visual analysis of the molecular binding events under an ordinary bright-field microscope and serve as a diffraction grating biosensor.}
}

% 19908395 
@Article{pmid19908395,
   Author="Davidson, E. A.  and VAN Blarcom, T.  and Levy, M.  and Ellington, A. D. ",
   Title="{{E}mulsion based selection of {T}7 promoters of varying activity}",
   Journal="Pac Symp Biocomput",
   Year="2010",
   Pages="433--443",
   Abstract={The ability to build and control complex biological systems is greatly enhanced by the generation of related parts with varying strengths. In this way, various parts can be strung together and the connectivity and expression levels can be matched for the desired system performance. Engineered gene circuits, both in vivo and in vitro, often utilize the T7 RNA polymerase in tandem with the T7 promoter for transcription. In this work, we describe the selection of T7 promoter variants of varying strength by emulsifying in vitro transcription with subsequent fluorescence activated cell sorting (FACS) to enrich for active promoters. Such variant promoters should be of use to synthetic biologists for both in vivo and in vitro applications.}
}

% 19816933 
@Article{pmid19816933,
   Author="Hall, B.  and Arshad, S.  and Seo, K.  and Bowman, C.  and Corley, M.  and Jhaveri, S. D.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization}",
   Journal="Curr Protoc Mol Biol",
   Year="2009",
   Volume="Chapter 24",
   Pages="Unit 24.3",
   Month="Oct",
   Abstract={This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially act as protein function inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate an ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.}
}

% 19816932 
@Article{pmid19816932,
   Author="Hall, B.  and Micheletti, J. M.  and Satya, P.  and Ogle, K.  and Pollard, J.  and Ellington, A. D. ",
   Title="{{D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection}",
   Journal="Curr Protoc Mol Biol",
   Year="2009",
   Volume="Chapter 24",
   Pages="Unit 24.2",
   Month="Oct",
   Abstract={Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer or via commercial synthesis companies. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.}
}

% 19776159 
@Article{pmid19776159,
   Author="Chen, X.  and Denison, L.  and Levy, M.  and Ellington, A. D. ",
   Title="{{D}irect selection for ribozyme cleavage activity in cells}",
   Journal="RNA",
   Year="2009",
   Volume="15",
   Number="11",
   Pages="2035--2045",
   Month="Nov",
   Abstract={Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments.}
}

% 19735583 
@Article{pmid19735583,
   Author="Hwang, T. W.  and Codrea, V.  and Ellington, A. D. ",
   Title="{{M}otifs from the deep}",
   Journal="J. Biol.",
   Year="2009",
   Volume="8",
   Number="8",
   Pages="72",
   Abstract={Because of the increasing recognition of the importance of non-coding RNAs in gene regulation, there is considerable interest in identifying RNA motifs in genomic data. In a recent report in BMC Genomics, Breaker and colleagues describe a new algorithm for identifying functional noncoding RNAs in metagenomic sequences of marine organisms, a strategy that may be particularly effective for discovering new and unique riboswitches.}
}

% 19575478 
@Article{pmid19575478,
   Author="Davidson, E. A.  and Dlugosz, P. J.  and Levy, M.  and Ellington, A. D. ",
   Title="{{D}irected evolution of proteins in vitro using compartmentalization in emulsions}",
   Journal="Curr Protoc Mol Biol",
   Year="2009",
   Volume="Chapter 24",
   Pages="Unit 24.6",
   Month="Jul",
   Abstract={This unit describes a protocol for the directed evolution of proteins utilizing in vitro compartmentalization. This method uses a large number of independent in vitro transcription and translation (IVTT) reactions in water droplets suspended in an oil emulsion to enable selection of proteins that bind a target molecule. Protein variants that bind the target also bind to and allow recovery of the genes that encoded them. This protocol serves as a basis for carrying out selections in emulsions, and can potentially be modified to select for other functionalities, including catalysis. This selection method is advantageous compared to alternative selection protocols due to the ability to screen through very large-size libraries and the ability to express and screen or select for functions that would otherwise be toxic or inaccessible to in vivo selections and screens.}
}

% 19563759 
@Article{pmid19563759,
   Author="Tabor, J. J.  and Salis, H. M.  and Simpson, Z. B.  and Chevalier, A. A.  and Levskaya, A.  and Marcotte, E. M.  and Voigt, C. A.  and Ellington, A. D. ",
   Title="{{A} synthetic genetic edge detection program}",
   Journal="Cell",
   Year="2009",
   Volume="137",
   Number="7",
   Pages="1272--1281",
   Month="Jun",
   Abstract={Edge detection is a signal processing algorithm common in artificial intelligence and image recognition programs. We have constructed a genetically encoded edge detection algorithm that programs an isogenic community of E. coli to sense an image of light, communicate to identify the light-dark edges, and visually present the result of the computation. The algorithm is implemented using multiple genetic circuits. An engineered light sensor enables cells to distinguish between light and dark regions. In the dark, cells produce a diffusible chemical signal that diffuses into light regions. Genetic logic gates are used so that only cells that sense light and the diffusible signal produce a positive output. A mathematical model constructed from first principles and parameterized with experimental measurements of the component circuits predicts the performance of the complete program. Quantitatively accurate models will facilitate the engineering of more complex biological behaviors and inform bottom-up studies of natural genetic regulatory networks.}
}

% 19502427 
@Article{pmid19502427,
   Author="Narayanaswamy, R.  and Levy, M.  and Tsechansky, M.  and Stovall, G. M.  and O'Connell, J. D.  and Mirrielees, J.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{W}idespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2009",
   Volume="106",
   Number="25",
   Pages="10147--10152",
   Month="Jun",
   Abstract={Proteins are likely to organize into complexes that assemble and disassemble depending on cellular needs. When approximately 800 yeast strains expressing GFP-tagged proteins were grown to stationary phase, a surprising number of proteins involved in intermediary metabolism and stress response were observed to form punctate cytoplasmic foci. The formation of these discrete physical structures was confirmed by immunofluorescence and mass spectrometry of untagged proteins. The purine biosynthetic enzyme Ade4-GFP formed foci in the absence of adenine, and cycling between punctate and diffuse phenotypes could be controlled by adenine subtraction and addition. Similarly, glutamine synthetase (Gln1-GFP) foci cycled reversibly in the absence and presence of glucose. The structures were neither targeted for vacuolar or autophagosome degradation nor colocalized with P bodies or major organelles. Thus, upon nutrient depletion we observe widespread protein assemblies displaying nutrient-specific formation and dissolution.}
}

% 19474083 
@Article{pmid19474083,
   Author="Simpson, Z. B.  and Tsai, T. L.  and Nguyen, N.  and Chen, X.  and Ellington, A. D. ",
   Title="{{M}odelling amorphous computations with transcription networks}",
   Journal="J R Soc Interface",
   Year="2009",
   Volume="6 Suppl 4",
   Pages="S523--533",
   Month="Aug",
   Abstract={The power of electronic computation is due in part to the development of modular gate structures that can be coupled to carry out sophisticated logical operations and whose performance can be readily modelled. However, the equivalences between electronic and biochemical operations are far from obvious. In order to help cross between these disciplines, we develop an analogy between complementary metal oxide semiconductor and transcriptional logic gates. We surmise that these transcriptional logic gates might prove to be useful in amorphous computations and model the abilities of immobilized gates to form patterns. Finally, to begin to implement these computations, we design unique hairpin transcriptional gates and then characterize these gates in a binary latch similar to that already demonstrated by Kim et al. (Kim, White & Winfree 2006 Mol. Syst. Biol. 2, 68 (doi:10.1038/msb4100099)). The hairpin transcriptional gates are uniquely suited to the design of a complementary NAND gate that can serve as an underlying basis of molecular computing that can output matter rather than electronic information.}
}

% 19431189 
@Article{pmid19431189,
   Author="Hall, B.  and Cater, S.  and Levy, M.  and Ellington, A. D. ",
   Title="{{K}inetic optimization of a protein-responsive aptamer beacon}",
   Journal="Biotechnol. Bioeng.",
   Year="2009",
   Volume="103",
   Number="6",
   Pages="1049--1059",
   Month="Aug",
   Abstract={Aptamers have been utilized as biosensors because they can be readily adapted to sensor platforms and signal transduction schemes through both rational design and selection. One highly generalizable scheme for the generation of the so-called aptamer beacons involves denaturing the aptamer with antisense oligonucleotides. For example, rational design methods have been utilized to adapt anti-thrombin aptamers to function as biosensors by hybridizing an antisense oligonucleotide containing a quencher to the aptamer containing a fluorescent label. In the presence of thrombin, the binding equilibrium is shifted, the antisense oligonucleotide dissociates, and the beacon lights up. By changing the affinity of the antisense oligonucleotide for the aptamer beacon, it has proven possible to change the extent of activation of the beacon. More importantly, modulating interactions between the antisense oligonucleotide and the aptamer strongly influences the kinetics of activation. Comparisons across multiple, designed aptamer beacons indicate that there is a strong inverse correlation between the thermodynamics of hybridization and the speed of activation, a finding that should prove to be generally useful in the design of future biosensors. By pre-organizing the thrombin-binding quadruplex within the aptamer the speed of response can be greatly increased. By integrating these various interactions, we were ultimately able to design aptamer beacons that were activated by threefold within 1 min of the addition of thrombin.}
}

% 19377997 
@Article{pmid19377997,
   Author="Piasecki, S. K.  and Hall, B.  and Ellington, A. D. ",
   Title="{{N}ucleic acid pool preparation and characterization}",
   Journal="Methods Mol. Biol.",
   Year="2009",
   Volume="535",
   Pages="3--18",
   Abstract={Random sequence nucleic acid pools can be used in a variety of applications, including the selection of functional nucleic acids such as protein binding sites, aptamers, and ribozymes. while the design, synthesis, and purification of pools is relatively straightforward, keeping track of the size and complexity of a nucleic acid pool can sometimes task even an experienced researcher. The following protocol takes the reader through the steps necessary for the preparation of a pool of known complexity.}
}

% 19295523 
@Article{pmid19295523,
   Author="Ellington, A. D. ",
   Title="{{B}ack to the future of nucleic acid self-amplification}",
   Journal="Nat. Chem. Biol.",
   Year="2009",
   Volume="5",
   Number="4",
   Pages="200--201",
   Month="Apr"
}

% 19271740 
@Article{pmid19271740,
   Author="Li, N.  and Ebright, J. N.  and Stovall, G. M.  and Chen, X.  and Nguyen, H. H.  and Singh, A.  and Syrett, A.  and Ellington, A. D. ",
   Title="{{T}echnical and biological issues relevant to cell typing with aptamers}",
   Journal="J. Proteome Res.",
   Year="2009",
   Volume="8",
   Number="5",
   Pages="2438--2448",
   Month="May",
   Abstract={A number of aptamers have been selected against cell surface biomarkers or against eukaryotic tissue culture cells themselves. To determine the general utility of aptamers for assessing the cell surface proteome, we developed a standardized flow cytometry assay and carried out a comprehensive study with 7 different aptamers and 14 different cell lines. By examining how aptamers performed with a variety of cell lines, we identified difficulties in using aptamers for cell typing. While there are some aptamers that show excellent correlation between cell surface binding and the expression of a biomarker on the cell surface, other aptamers showed nonspecific binding by flow cytometry. For example, it has recently been claimed that an anti-PTK7 (protein tyrosine kinase 7) aptamer identified a new biomarker for leukemia cells, but data with the additional cell lines shows that it is possible that the aptamer instead identifies a propensity for adherence. Better understanding and controlling for the role of background and nonspecific binding to cells should open the way to using arrays of aptamers for describing and quantifying the cell surface proteome.}
}

% 19177559 
@Article{pmid19177559,
   Author="Seo, M. J.  and Jeong, K. J.  and Leysath, C. E.  and Ellington, A. D.  and Iverson, B. L.  and Georgiou, G. ",
   Title="{{E}ngineering antibody fragments to fold in the absence of disulfide bonds}",
   Journal="Protein Sci.",
   Year="2009",
   Volume="18",
   Number="2",
   Pages="259--267",
   Month="Feb",
   Abstract={Disulfide bonds play a critical role in the stabilization of the immunoglobulin beta-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193-9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues.}
}

% 19159107 
@Article{pmid19159107,
   Author="Pai, S. S.  and Ellington, A. D. ",
   Title="{{U}sing {R}{N}{A} aptamers and the proximity ligation assay for the detection of cell surface antigens}",
   Journal="Methods Mol. Biol.",
   Year="2009",
   Volume="504",
   Pages="385--398",
   Abstract={The detection and typing of tumor cells based on differentially or similarly expressed antigens (biomarkers) have proven to be increasingly important for the diagnosis and treatment of various cancers. Sensitive techniques for the detection of cell surface antigens are therefore crucial for the early and accurate detection of cancer. Although techniques such as ELISA and tissue staining have proven their worth, these techniques often either require substantial amounts of starting material or are prone to high background and false negatives. The proximity ligation assay (PLA) has proven to be an exquisitely sensitive technique with very low background. Two probes that bind adjacent to one another on a protein target can be ligated, yielding a unique amplicon that can be sensitively detected by real-time PCR. We have now adapted PLA to cell surface protein targets using modified RNA aptamers, and have shown that aptamer-based cell surface PLA can successfully detect and differentiate between cells that differentially express a tumor antigen, the prostate specific membrane antigen (PSMA).}
}

% 19121689 
@Article{pmid19121689,
   Author="Gnanam, A. J.  and Hall, B.  and Shen, X.  and Piasecki, S.  and Vernados, A.  and Galyov, E. E.  and Smither, S. J.  and Kitto, G. B.  and Titball, R. W.  and Ellington, A. D.  and Brown, K. A. ",
   Title="{{D}evelopment of aptamers specific for potential diagnostic targets in {B}urkholderia pseudomallei}",
   Journal="Trans. R. Soc. Trop. Med. Hyg.",
   Year="2008",
   Volume="102 Suppl 1",
   Pages="S55--57",
   Month="Dec",
   Abstract={Improved diagnostic reagents would be of considerable benefit in enhancing the specificity and sensitivity of rapid assays for Burkholderia pseudomallei, the causative agent of melioidosis. The purpose of this work is to develop aptamers, high affinity RNA-based molecular recognition molecules, which could be used as reagents for identification of the whole organism in assays of biological samples. Data are presented demonstrating the purification of recombinant B. pseudomallei secreted or surface-exposed macromolecules, which have been expressed in Escherichia coli, and the initial stages of aptamer generation using these recombinant proteins. Future studies will focus upon the expansion of this methodology to include other target macromolecules located on or near the outer membrane of this organism.}
}

% 19053807 
@Article{pmid19053807,
   Author="Narayanaswamy, R.  and Moradi, E. K.  and Niu, W.  and Hart, G. T.  and Davis, M.  and McGary, K. L.  and Ellington, A. D.  and Marcotte, E. M. ",
   Title="{{S}ystematic definition of protein constituents along the major polarization axis reveals an adaptive reuse of the polarization machinery in pheromone-treated budding yeast}",
   Journal="J. Proteome Res.",
   Year="2009",
   Volume="8",
   Number="1",
   Pages="6--19",
   Month="Jan",
   Abstract={Polarizing cells extensively restructure cellular components in a spatially and temporally coupled manner along the major axis of cellular extension. Budding yeast are a useful model of polarized growth, helping to define many molecular components of this conserved process. Besides budding, yeast cells also differentiate upon treatment with pheromone from the opposite mating type, forming a mating projection (the 'shmoo') by directional restructuring of the cytoskeleton, localized vesicular transport and overall reorganization of the cytosol. To characterize the proteomic localization changes accompanying polarized growth, we developed and implemented a novel cell microarray-based imaging assay for measuring the spatial redistribution of a large fraction of the yeast proteome, and applied this assay to identify proteins localized along the mating projection following pheromone treatment. We further trained a machine learning algorithm to refine the cell imaging screen, identifying additional shmoo-localized proteins. In all, we identified 74 proteins that specifically localize to the mating projection, including previously uncharacterized proteins (Ycr043c, Ydr348c, Yer071c, Ymr295c, and Yor304c-a) and known polarization complexes such as the exocyst. Functional analysis of these proteins, coupled with quantitative analysis of individual organelle movements during shmoo formation, suggests a model in which the basic machinery for cell polarization is generally conserved between processes forming the bud and the shmoo, with a distinct subset of proteins used only for shmoo formation. The net effect is a defined ordering of major organelles along the polarization axis, with specific proteins implicated at the proximal growth tip.}
}

% 23496781 
@Article{pmid23496781,
   Author="Pai, S.  and Roberts, A.  and Ellington, A. D. ",
   Title="{{A}ptamer amplification: divide and signal}",
   Journal="Expert Opin Med Diagn",
   Year="2008",
   Volume="2",
   Number="12",
   Pages="1333--1346",
   Month="Dec",
   Abstract={Aptamers are selected nucleic acids that bind their targets with affinities and specificities that are often comparable to those of monoclonal antibodies.\\ Although aptamers have been adapted to a wide variety of assay formats, the fact that they are nucleic acids makes them uniquely useful for adaptation to amplification assays. Aptamer-based amplification assays have not previously been reviewed separately.\\ Aptamers can be used as simple binding reagents and then detected by methods such as the polymerase chain reaction and the rolling circle amplification assay. In addition, though, aptamers can undergo programmed, ligand-dependent conformational changes, or can form unique quarternary structures that lead to amplification.\\ Analytical assays that involve aptamers and nuleic acid amplification technologies can be used to detect sensitively target proteins, often in the nanomolar or picomolar range. However, in many cases there are no obvious advantages to amplification assays relative to other assay formats. In all likelihood all formats are limited by the dissociation constants of the aptamers themselves. The one exception to this is the proximity ligation assay, where sequence amplification allows the detection of extremely small quantities of ligands relative to background.}
}

% 19009652 
@Article{pmid19009652,
   Author="Fan, S.  and Wu, F.  and Martiniuk, F.  and Hale, M. L.  and Ellington, A. D.  and Tchou-Wong, K. M. ",
   Title="{{P}rotective effects of anti-ricin {A}-chain {R}{N}{A} aptamer against ricin toxicity}",
   Journal="World J. Gastroenterol.",
   Year="2008",
   Volume="14",
   Number="41",
   Pages="6360--6365",
   Month="Nov",
   Abstract={To investigate the therapeutic potential of an RNA ligand (aptamer) specific for the catalytic ricin A-chain (RTA), the protective effects of a 31-nucleotide RNA aptamer (31RA), which formed a high affinity complex with RTA, against ricin-induced toxicity in cell-based luciferase translation and cell cytotoxicity assays were evaluated.\\ To test the therapeutic potential of anti-RTA aptamers in Chinese hamster ovary (CHO) AA8 cells stably transfected with a tetracycline regulatable promoter, ricin ribotoxicity was measured using luciferase and ricin-induced cytotoxicity was ascertained by MTS cell proliferation assay with tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium].\\ Inhibition of protein synthesis by ricin in CHO AA8 cells resulted in diminished luciferase activity and treatment with polyclonal antibody against deglycosylated RTA (dgA) neutralized the inhibitory effects of ricin on luciferase activity and protected against ricin-induced cytotoxicity as measured by MTS assay. The 31RA anti-RTA aptamer inhibited the translation of luciferase mRNA in cell-free reticulocyte translation assay. 31RA aptamer also partially neutralized the inhibitory effects of ricin on luciferase activity and partially protected against ricin-induced cytotoxicity in CHO AA8 cells.\\ We have shown that anti-RTA RNA aptamer can protect against ricin ribotoxicity in cell-based luciferase and cell cytotoxicity assays. Hence, RNA aptamer that inhibits RTA enzymatic activity represents a novel class of nucleic acid inhibitor that has the potential to be developed as a therapeutic agent for the treatment of ricin intoxication.}
}

% 19008359 
@Article{pmid19008359,
   Author="Paukstelis, P. J.  and Ellington, A. D. ",
   Title="{{R}olling out {D}{N}{A} nanostructures in vivo}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2008",
   Volume="105",
   Number="46",
   Pages="17593--17594",
   Month="Nov"
}

% 18948292 
@Article{pmid18948292,
   Author="Li, N.  and Wang, Y.  and Pothukuchy, A.  and Syrett, A.  and Husain, N.  and Gopalakrisha, S.  and Kosaraju, P.  and Ellington, A. D. ",
   Title="{{A}ptamers that recognize drug-resistant {H}{I}{V}-1 reverse transcriptase}",
   Journal="Nucleic Acids Res.",
   Year="2008",
   Volume="36",
   Number="21",
   Pages="6739--6751",
   Month="Dec",
   Abstract={Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.}
}

% 18804035 
@Article{pmid18804035,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{D}irected evolution of streptavidin variants using in vitro compartmentalization}",
   Journal="Chem. Biol.",
   Year="2008",
   Volume="15",
   Number="9",
   Pages="979--989",
   Month="Sep",
   Abstract={We have developed and implemented an in vitro compartmentalization (IVC) selection scheme for the identification of streptavidin (SA) variants with altered specificities for the biotin analog desthiobiotin. Wild-type SA and selected variants bind desthiobiotin with similar affinities (approximately 10(-13) M), but the variants have off rates almost 50 times slower and a half-life for dissociation of 24 hr at 25 degrees C. The utility of streptavidin variants with altered specificities and kinetic properties was shown by constructing protein microarrays that could be used to differentially organize and immobilize DNAs bearing these ligands. The methods we have developed should prove to be generally useful for generating a variety of novel SA reagents and for evolving other extremely high-affinity protein:ligand couples.}
}

% 18775793 
@Article{pmid18775793,
   Author="Ellington, A. D.  and Chen, X.  and Robertson, M.  and Syrett, A. ",
   Title="{{E}volutionary origins and directed evolution of {R}{N}{A}}",
   Journal="Int. J. Biochem. Cell Biol.",
   Year="2009",
   Volume="41",
   Number="2",
   Pages="254--265",
   Month="Feb",
   Abstract={In vitro selection experiments show first and foremost that it is possible that functional nucleic acids can arise from random sequence libraries. Indeed, even simple sequence and structural motifs can prove to be robust binding species and catalysts, indicating that it may have been possible to transition from even the earliest self-replicators to a nascent, RNA-catalyzed metabolism. Because of the diversity of aptamers and ribozymes that can be selected, it is possible to construct a 'fossil record' of the evolution of the RNA world, with in vitro selected catalysts filling in as doppelgangers for molecules long gone. In this way a plausible pathway from simple oligonucleotide replicators to genomic polymerases can be imagined, as can a pathway from basal ribozyme activities to the ribosome. Most importantly, though, in vitro selection experiments can give a true and quantitative idea of the likelihood that these scenarios could have played out in the RNA world. Simple binding species and catalysts could have evolved into other structures and functions. As replicating sequences grew longer, new, more complex functions or faster catalytic activities could have been accessed. Some activities may have been isolated in sequence space, but others could have been approached along large, interconnected neutral networks. As the number, type, and length of ribozymes increased, RNA genomes would have evolved and eventually there would have been no area in a fitness landscape that would have been inaccessible. Self-replication would have inexorably led to life.}
}

% 18594898 
@Article{pmid18594898,
   Author="Cowperthwaite, M. C.  and Ellington, A. D. ",
   Title="{{B}ioinformatic analysis of the contribution of primer sequences to aptamer structures}",
   Journal="J. Mol. Evol.",
   Year="2008",
   Volume="67",
   Number="1",
   Pages="95--102",
   Month="Jul",
   Abstract={Aptamers are nucleic acid molecules selected in vitro to bind a particular ligand. While numerous experimental studies have examined the sequences, structures, and functions of individual aptamers, considerably fewer studies have applied bioinformatics approaches to try to infer more general principles from these individual studies. We have used a large Aptamer Database to parse the contributions of both random and constant regions to the secondary structures of more than 2000 aptamers. We find that the constant, primer-binding regions do not, in general, contribute significantly to aptamer structures. These results suggest that (a) binding function is not contributed to nor constrained by constant regions; (b) in consequence, the landscape of functional binding sequences is sparse but robust, favoring scenarios for short, functional nucleic acid sequences near origins; and (c) many pool designs for the selection of aptamers are likely to prove robust.}
}

% 18563250 
@Article{pmid18563250,
   Author="Tabor, J. J.  and Bayer, T. S.  and Simpson, Z. B.  and Levy, M.  and Ellington, A. D. ",
   Title="{{E}ngineering stochasticity in gene expression}",
   Journal="Mol Biosyst",
   Year="2008",
   Volume="4",
   Number="7",
   Pages="754--761",
   Month="Jul",
   Abstract={Stochastic fluctuations (noise) in gene expression can cause members of otherwise genetically identical populations to display drastically different phenotypes. An understanding of the sources of noise and the strategies cells employ to function reliably despite noise is proving to be increasingly important in describing the behavior of natural organisms and will be essential for the engineering of synthetic biological systems. Here we describe the design of synthetic constructs, termed ribosome competing RNAs (rcRNAs), as a means to rationally perturb noise in cellular gene expression. We find that noise in gene expression increases in a manner proportional to the ability of an rcRNA to compete for the cellular ribosome pool. We then demonstrate that operons significantly buffer noise between coexpressed genes in a natural cellular background and can even reduce the level of rcRNA enhanced noise. These results demonstrate that synthetic genetic constructs can significantly affect the noise profile of a living cell and, importantly, that operons are a facile genetic strategy for buffering against noise.}
}

% 18541130 
@Article{pmid18541130,
   Author="Yang, L.  and Ellington, A. D. ",
   Title="{{R}eal-time {P}{C}{R} detection of protein analytes with conformation-switching aptamers}",
   Journal="Anal. Biochem.",
   Year="2008",
   Volume="380",
   Number="2",
   Pages="164--173",
   Month="Sep",
   Abstract={We have developed a novel method that uses conformation-switching aptamers for real-time PCR analysis of protein analytes. The aptamers have been designed so that they assume one secondary structure in the absence of a protein analyte and a different secondary structure in the presence of a protein such as thrombin or platelet-derived growth factor (PDGF). The protein-bound structure in turn assembles a ligation junction for the addition of a real-time PCR primer. Protein concentrations could be specifically detected into the picomolar range, even in the presence of cell lysates. The method has advantages relative to both immunoPCR (because no signal is produced by background binding) and the proximity ligation assay (PLA) (because only one epitope, rather than two epitopes, on a protein surface must be bound).}
}

% 18507505 
@Article{pmid18507505,
   Author="Ellington, A. D. ",
   Title="{{M}an versus machine versus ribozyme}",
   Journal="PLoS Biol.",
   Year="2008",
   Volume="6",
   Number="5",
   Pages="e132",
   Month="May"
}

% 18476324 
@Article{pmid18476324,
   Author="Tabor, J. J.  and Davidson, E. A.  and Ellington, A. D. ",
   Title="{{D}eveloping {R}{N}{A} tools for engineered regulatory systems}",
   Journal="Biotechnol. Genet. Eng. Rev.",
   Year="2006",
   Volume="22",
   Pages="21--44"
}

% 18428900 
@Article{pmid18428900,
   Author="Knudsen, S. M.  and Robertson, M. P.  and Ellington, A. D. ",
   Title="{{I}n vitro selection using modified or unnatural nucleotides}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2002",
   Volume="Chapter 9",
   Pages="Unit 9.6",
   Month="Feb",
   Abstract={The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.}
}

% 18428881 
@Article{pmid18428881,
   Author="Jhaveri, S. D.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2001",
   Volume="Chapter 9",
   Pages="Unit 9.3",
   Month="May",
   Abstract={This unit describes the selection of aptamers from a pool of single-stranded RNA by binding to a protein target. Aptamers generated from this selection experiment can potentially function as protein inhibitors, and may find applications as therapeutic or diagnostic reagents. A pool of dsDNA is used to generate a ssRNA pool, which is mixed with the protein target. Bound complexes are separated from unbound reagents by filtration, and the RNA:protein complexes are amplified by a combination of reverse transcription, PCR, and in vitro transcription.}
}

% 18428880 
@Article{pmid18428880,
   Author="Pollard, J.  and Bell, S. D.  and Ellington, A. D. ",
   Title="{{D}esign, synthesis, and amplification of {D}{N}{A} pools for in vitro selection}",
   Journal="Curr Protoc Nucleic Acid Chem",
   Year="2001",
   Volume="Chapter 9",
   Pages="Unit 9.2",
   Month="May",
   Abstract={Preparation of a random-sequence DNA pool is presented. The degree of randomization and the length of the random sequence are discussed, as is synthesis of the pool using a DNA synthesizer. Purification of a single-stranded pool and conversion to a double-stranded pool are presented as step-by-step protocols. Support protocols describe determination of the complexity and skewing of the pool, and optimization of amplification conditions.}
}

% 18398956 
@Article{pmid18398956,
   Author="Ellington, A. D. ",
   Title="{{Q} &{A}. {A}ndrew {D}. {E}llington}",
   Journal="Curr. Biol.",
   Year="2008",
   Volume="18",
   Number="5",
   Pages="R184--185",
   Month="Mar"
}

% 18265210 
@Article{pmid18265210,
   Author="Jhaveri, S. D.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} aptamers to a protein target by filter immobilization}",
   Journal="Curr Protoc Mol Biol",
   Year="2001",
   Volume="Chapter 24",
   Pages="Unit 24.3",
   Month="May",
   Abstract={In vitro selection of RNA aptamers that bind to a protein target is detailed in the protocols presented in this unit. Aptamers generated from these types of selection experiments can potentially function as protein inhibitors, and are often used as diagnostic or therapeutic reagents.}
}

% 18265209 
@Article{pmid18265209,
   Author="Pollard, J.  and Bell, S. D.  and Ellington, A. D. ",
   Title="{{D}esign, synthesis, and amplification of {D}{N}{A} pools for construction of combinatorial pools and libraries}",
   Journal="Curr Protoc Mol Biol",
   Year="2001",
   Volume="Chapter 24",
   Pages="Unit 24.2",
   Month="May",
   Abstract={This unit describes the design, synthesis, and amplification of random-sequence DNA pools, from which functional nucleic acid-binding or catalytic species can be selected. Since it is an expensive and time-consuming process, the authors have provided an extensive strategic planning section to guide investigators in designing and constructing the pool.}
}

% 18049815 
@Article{pmid18049815,
   Author="Rajendran, M.  and Ellington, A. D. ",
   Title="{{S}election of fluorescent aptamer beacons that light up in the presence of zinc}",
   Journal="Anal Bioanal Chem",
   Year="2008",
   Volume="390",
   Number="4",
   Pages="1067--1075",
   Month="Feb",
   Abstract={In order to generate nucleic acid biosensors that could undergo a reversible conformation change in the presence of the metal zinc, a random sequence pool of single-stranded DNA was immobilized on an oligonucleotide affinity column. In the presence of zinc, those species that underwent a conformational change were released from the column, collected, and amplified. A series of negative and positive selections refined the metal specificity of the selected aptamer beacons. Since the aptamer beacons contained a fluorophore, while the bound oligonucleotide contained a quencher, zinc binding also resulted in an increase in fluorescence. One of the selected beacons, Zn-6m2, bound zinc in the low micromolar range, gave a dose-dependent fluorescence signal, and showed an approximately sixfold increase in fluorescence on zinc binding. While some cross-reactivity with cadmium was observed, it should nonetheless prove possible to use the novel selection method to generate and tune the specificity of a variety of reversible metal biosensors. Such biosensors could potentially be used for continuous monitoring of metals in environmental samples.}
}

% 17723365 
@Article{pmid17723365,
   Author="Cho, E. J.  and Collett, J. R.  and Szafranska, A. E.  and Ellington, A. D. ",
   Title="{{O}ptimization of aptamer microarray technology for multiple protein targets}",
   Journal="Anal. Chim. Acta",
   Year="2006",
   Volume="564",
   Number="1",
   Pages="82--90",
   Month="Mar",
   Abstract={Aptamer-based microarrays for the quantitation of multiple protein analytes have been developed. A multiplex aptamer microarray was generated by printing two RNA aptamers (anti-lysozyme and anti-ricin) and two DNA aptamers (anti-IgE and anti-thrombin) on to either streptavidin (SA) or neutravidin (NA)-coated glass slides. However, substantial optimization was required in order to ensure the simultaneous function of the aptamer:analyte pairs. The effects of protein labeling, assay buffer, surface coating, and immobilization chemistry and orientation were investigated. A single buffer (PBS buffer containing 5 mM MgCl2 and 0.1% Tween 20) was found to work well with all the aptamers, even though this was not the buffer originally used in their selection, while neutravidin-coated slides yielded a lower detection limit, wider detection range, and more uniform background than streptavidin-coated slides. Incubation with Cy3-labeled proteins yielded sensitive, target-specific, and dose-dependent responses to each protein. Target protein concentrations as low as 72 pg/mL (5 pM, lysozyme), 15 ng/mL (0.5 nM, ricin), 1.9 ng/mL (0.01 nM, IgE), and 170 ng/mL (5 nM, thrombin) could be detected. These results show that aptamer arrays can potentially be used with numerous proteins in parallel, furthering the notion that aptamer arrays may be useful in proteomics.}
}

% 17649966 
@Article{pmid17649966,
   Author="Ellington, A. D. ",
   Title="{{W}hat's so great about {R}{N}{A}?}",
   Journal="ACS Chem. Biol.",
   Year="2007",
   Volume="2",
   Number="7",
   Pages="445--448",
   Month="Jul"
}

% 17458167 
@Article{pmid17458167,
   Author="Chu, T.  and Ebright, J.  and Ellington, A. D. ",
   Title="{{U}sing aptamers to identify and enter cells}",
   Journal="Curr. Opin. Mol. Ther.",
   Year="2007",
   Volume="9",
   Number="2",
   Pages="137--144",
   Month="Apr",
   Abstract={While nucleic acid binding species (aptamers) have previously been selected against a variety of biomedically important protein targets, the selection of aptamers against complex targets, such as cell surfaces and organisms, is becoming increasingly feasible. A number of aptamers have now been generated that can target specific cell-surface antigens and cell types, including tumor biomarkers and tumor cells. The generation of anti-cell aptamers has important implications for identifying disease-specific biomarkers, generating diagnostics, and developing novel drug delivery strategies.}
}

% 17443876 
@Article{pmid17443876,
   Author="Chen, X.  and Li, N.  and Ellington, A. D. ",
   Title="{{R}ibozyme catalysis of metabolism in the {R}{N}{A} world}",
   Journal="Chem. Biodivers.",
   Year="2007",
   Volume="4",
   Number="4",
   Pages="633--655",
   Month="Apr",
   Abstract={In vitro selection has proven to be a useful means of explore the molecules and catalysts that may have existed in a primordial 'RNA world'. By selecting binding species (aptamers) and catalysts (ribozymes) from random sequence pools, the relationship between biopolymer complexity and function can be better understood, and potential evolutionary transitions between functional molecules can be charted. In this review, we have focused on several critical events or transitions in the putative RNA world: RNA self-replication; the synthesis and utilization of nucleotide-based cofactors; acyl-transfer reactions leading to peptide and protein synthesis; and the basic metabolic pathways that are found in modern living systems.}
}

% 17378540 
@Article{pmid17378540,
   Author="Yang, L.  and Fung, C. W.  and Cho, E. J.  and Ellington, A. D. ",
   Title="{{R}eal-time rolling circle amplification for protein detection}",
   Journal="Anal. Chem.",
   Year="2007",
   Volume="79",
   Number="9",
   Pages="3320--3329",
   Month="May",
   Abstract={Real-time nucleic acid amplification methods can be extremely useful for the identification and quantitation of nucleic acid analytes, but are more difficult to adapt to protein or other analytes. To facilitate the development of real-time rolling circle amplification (RCA) for protein targets, we have developed a novel type of conformation-switching aptamer that can be circularized upon interaction with its protein target, the platelet-derived growth factor (PDGF). Using the structure-switching aptamer, real-time RCA can be used to specifically quantitate PDGF down to the low-nanomolar range (limit of detection, 0.4 nM), even against a background of cellular lysate. The aptamer can also be adapted to RCA on surfaces, although quantitation proved to be more difficult. One of the great advantages of the method described herein is that it can be immediately adapted to almost any aptamer and does not require two or more affinity reagents as do sandwich or proximity assays.}
}

% 17173026 
@Article{pmid17173026,
   Author="Davidson, E. A.  and Ellington, A. D. ",
   Title="{{S}ynthetic {R}{N}{A} circuits}",
   Journal="Nat. Chem. Biol.",
   Year="2007",
   Volume="3",
   Number="1",
   Pages="23--28",
   Month="Jan",
   Abstract={Natural and engineered RNA 'parts' can perform a variety of functions, including hybridizing to targets, binding ligands and undergoing programmed conformational changes, and catalyzing reactions. These RNA parts can in turn be assembled into synthetic genetic circuits that regulate gene expression by acting either in cis or in trans on mRNAs. As more parts are discovered and engineered, it should be increasingly possible to create synthetic RNA circuits that are able to carry out complex logical operations in cells, either superimposed on or autonomous to extant gene regulation.}
}

% 17165697 
@Article{pmid17165697,
   Author="Knudsen, S. M.  and Lee, J.  and Ellington, A. D.  and Savran, C. A. ",
   Title="{{R}ibozyme-mediated signal augmentation on a mass-sensitive biosensor}",
   Journal="J. Am. Chem. Soc.",
   Year="2006",
   Volume="128",
   Number="50",
   Pages="15936--15937",
   Month="Dec",
   Abstract={Mass-based detection methods such as the quartz crystal microbalance (QCM) offer an attractive option to label-based methods; however the sensitivity is generally lower by comparison. In particular, low-molecular-weight analytes can be difficult to detect based on mass addition alone. In this communication, we present the use of effector-dependent ribozymes (aptazymes) as reagents for augmenting small ligand detection on a mass-sensitive device. Two distinct aptazymes were chosen: an L1-ligase-based aptazyme (L1-Rev), which is activated by a small peptide (MW approximately 2.4 kDa) from the HIV-1 Rev protein, and a hammerhead cleavase-based aptazyme (HH-theo3) activated by theophylline (MW = 180 Da). Aptazyme activity was observed in real time, and low-molecular-weight analyte detection has been successfully demonstrated with both aptazymes.}
}

% 17041257 
@Article{pmid17041257,
   Author="Bacher, J. M.  and Ellington, A. D. ",
   Title="{{G}lobal incorporation of unnatural amino acids in {E}scherichia coli}",
   Journal="Methods Mol. Biol.",
   Year="2007",
   Volume="352",
   Pages="23--34",
   Abstract={The incorporation of amino acid analogs is becoming increasingly useful. Site-specific incorporation of unnatural amino acids allows the application of chemical biology to protein-specific investigations and applications. However, the global incorporation of unnatural amino acids allows for tests of proteomic and genetic code hypotheses. For example, the adaptation of organisms to unnatural amino acids may lead to new genetic codes. To understand and quantify changes from such perturbations, an understanding is required of the microbiological and proteomic responses to the incorporation of unnatural amino acids. Here we describe protocols to characterize the effects of such proteome-wide perturbations.}
}

% 16996258 
@Article{pmid16996258,
   Author="Hall, B.  and Hesselberth, J. R.  and Ellington, A. D. ",
   Title="{{C}omputational selection of nucleic acid biosensors via a slip structure model}",
   Journal="Biosens Bioelectron",
   Year="2007",
   Volume="22",
   Number="9-10",
   Pages="1939--1947",
   Month="Apr",
   Abstract={Aptamers have been shown to undergo ligand-dependent conformational changes, and can be joined to ribozymes to create allosteric ribozymes (aptazymes). An anti-flavin (FMN) aptamer joined to the hammerhead ribozyme yielded an aptazyme that underwent small, FMN-dependent displacements in the helix that joined the aptamer and ribozyme. This 'slip structure' model in which alternative sets of base-pairs are formed in the absence and presence of ligand proved amenable to energetic and computational modeling. Initial successes in modeling the activities of known aptazymes led to the in silico selection of new ligand-dependent aptazymes from virtual pools that contained millions of members. Those aptazymes that were predicted to best fit the slip structure model were synthesized and assayed, and the best-designed aptazyme was activated 60-fold by FMN. The slip structure model proved to be generalizable, and could be applied with equal facility to computationally generate aptazymes that proved to be experimentally activated by other ligands (theophylline) or that contained other catalytic cores (hairpin ribozyme). Moreover, the slip structure model could be applied to the prediction of a ligand-dependent aptamer beacon biosensor in which the addition of the protein vascular endothelial growth factor (VegF) led to a 10-fold increase in fluorescent signal.}
}

% 16834439 
@Article{pmid16834439,
   Author="Garibotti, A. V.  and Knudsen, S. M.  and Ellington, A. D.  and Seeman, N. C. ",
   Title="{{F}unctional {D}{N}{A}zymes organized into two-dimensional arrays}",
   Journal="Nano Lett.",
   Year="2006",
   Volume="6",
   Number="7",
   Pages="1505--1507",
   Month="Jul",
   Abstract={DNAzymes are catalytically active DNA molecules, which have previously been described in solution. Here, we organize these molecules into a series of two-dimensional (2D) arrays using a periodic arrangement of DNA structures based on the DNA double crossover motif. We demonstrate by means of atomic force microscopy that the DNAzymes are organized according to the design and that they retain their activity when attached in linear strings within the context of the 2D array.}
}

% 16778167 
@Article{pmid16778167,
   Author="Chu, T. C.  and Marks, J. W.  and Lavery, L. A.  and Faulkner, S.  and Rosenblum, M. G.  and Ellington, A. D.  and Levy, M. ",
   Title="{{A}ptamer:toxin conjugates that specifically target prostate tumor cells}",
   Journal="Cancer Res.",
   Year="2006",
   Volume="66",
   Number="12",
   Pages="5989--5992",
   Month="Jun",
   Abstract={We have used RNA aptamer:gelonin conjugates to target and specifically destroy cells overexpressing the known cancer biomarker prostate-specific membrane antigen (PSMA). Aptamer:toxin conjugates have an IC50 of 27 nmol/L and display an increased potency of at least 600-fold relative to cells that do not express PSMA. The aptamer not only promotes uptake into target cells but also decreases the toxicity of gelonin in non-target cells. These results validate the notion that "escort aptamers" may be useful for the treatment of specific tumors expressing unique antigen targets.}
}

% 16740739 
@Article{pmid16740739,
   Author="Chu, T. C.  and Twu, K. Y.  and Ellington, A. D.  and Levy, M. ",
   Title="{{A}ptamer mediated si{R}{N}{A} delivery}",
   Journal="Nucleic Acids Res.",
   Year="2006",
   Volume="34",
   Number="10",
   Pages="e73",
   Abstract={Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.}
}

% 16713750 
@Article{pmid16713750,
   Author="Cho, E. J.  and Ellington, A. D. ",
   Title="{{O}ptimization of the biological component of a bioelectrochemical cell}",
   Journal="Bioelectrochemistry",
   Year="2007",
   Volume="70",
   Number="1",
   Pages="165--172",
   Month="Jan",
   Abstract={The efficiency of electron transduction by Shewanella oneidensis MR-1 was investigated both in batch culture and in a dual-chambered electrochemical cell. Aerobically grown bacteria were inoculated into an insoluble FeOOH suspension in an anaerobic environment. As the bacteria reduced Fe(III) to Fe(II) there was a visible color change from red to bluish black; this simple color change assay proved to be a robust method for determining the electrochemical activity of S. oneidensis MR-1. In an effort to improve electricity production by S. oneidensis MR-1, the performance of the electrochemical cell with lactate as a fuel was first optimized with respect to both poised potential and fuel concentration. Ultimately, electricity production proved to be proportional to both the poised potential and to fuel concentration. In particular, higher poised potentials increased charge production. Finally, we attempted to optimize the bacteria themselves for the efficient transduction of reduced carbon sources into electricity. The batch culture underwent a series of serial dilutions; after the 4th dilution the microbe population exhibited a 30% increase in charge production. We are further exploring whether this increase was due to metabolic adaptations or to genetic mutations, and examining additional ways to evolve electrogenic organisms.}
}

% 16701231 
@Article{pmid16701231,
   Author="Bacher, J. M.  and Hughes, R. A.  and Tze-Fei Wong, J.  and Ellington, A. D. ",
   Title="{{E}volving new genetic codes}",
   Journal="Trends Ecol. Evol. (Amst.)",
   Year="2004",
   Volume="19",
   Number="2",
   Pages="69--75",
   Month="Feb",
   Abstract={Although the genetic code is almost universal, natural variations exist that have caused evolutionary biologists to speculate about codon evolution. There are two predominant hypotheses that specify either a gradual (ambiguous intermediate) or stochastic (codon capture) change in the code. These hypotheses are similar to two biotechnology techniques that have been used to engineer the genetic code: a 'top down' approach, in which the whole organism is evolved for the ability to incorporate unnatural amino acids, and a 'bottom up' approach, in which aminoacyl-tRNA synthetases and their cognate tRNAs are engineered. The biotechnology experiments provide insights into natural codon evolution, and a combination of these approaches should enable the evolution of organisms that can incorporate unnatural amino acids throughout their proteomes.}
}

% 16648360 
@Article{pmid16648360,
   Author="Tabor, J. J.  and Levy, M.  and Ellington, A. D. ",
   Title="{{D}eoxyribozymes that recode sequence information}",
   Journal="Nucleic Acids Res.",
   Year="2006",
   Volume="34",
   Number="8",
   Pages="2166--2172",
   Abstract={Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can 'read' one sequence and then 'write' (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs.}
}

% 16621675 
@Article{pmid16621675,
   Author="Lee, J. F.  and Stovall, G. M.  and Ellington, A. D. ",
   Title="{{A}ptamer therapeutics advance}",
   Journal="Curr Opin Chem Biol",
   Year="2006",
   Volume="10",
   Number="3",
   Pages="282--289",
   Month="Jun",
   Abstract={Aptamers are selected nucleic acid binding species with affinities and specificities for protein targets that rival those of monoclonal antibodies. Furthermore, aptamers have definite advantages over antibodies, in that they can be chemically synthesized and modifications can be introduced that improve their stabilities and pharmacokinetic properties. A number of aptamers against therapeutically important targets have shown efficacy in cell and animal models, and a handful of aptamers are now in clinical trials or are being used as drugs. Recent advances in selection technologies and a more thorough exploration of how to deliver nucleic acids to target cells and tissues should further speed the process of drug development.}
}

% 16607663 
@Article{pmid16607663,
   Author="Schmid, M. J.  and Manthiram, K.  and Grayson, S. M.  and Willson, J. C.  and Meiring, J. E.  and Bell, K. M.  and Ellington, A. D.  and Willson, C. G. ",
   Title="{{F}eature multiplexing--improving the efficiency of microarray devices}",
   Journal="Angew. Chem. Int. Ed. Engl.",
   Year="2006",
   Volume="45",
   Number="20",
   Pages="3338--3341",
   Month="May"
}

% 16594629 
@Article{pmid16594629,
   Author="Narayanaswamy, R.  and Ellington, A. D. ",
   Title="{{E}ngineering {R}{N}{A}-based circuits}",
   Journal="Handb Exp Pharmacol",
   Year="2006",
   Number="173",
   Pages="423--445",
   Abstract={Nucleic acids can modulate gene function by base-pairing, via the molecular recognition of proteins and metabolites, and by catalysis. This diversity of functions can be combined with the ability to engineer nucleic acids based on Watson-Crick base-pairing rules to create a modular set of molecular "tools" for biotechnological and medical interventions in cellular metabolism. However, these individual RNA-based tools are most powerful when combined into rational logical or regulatory circuits, and the circuits can in turn be evolved for optimal function. Examples of genetic circuits that control translation and transcription are herein detailed, and more complex circuits with medical applications are anticipated.}
}

% 16507139 
@Article{pmid16507139,
   Author="Narayanaswamy, R.  and Niu, W.  and Scouras, A. D.  and Hart, G. T.  and Davies, J.  and Ellington, A. D.  and Iyer, V. R.  and Marcotte, E. M. ",
   Title="{{S}ystematic profiling of cellular phenotypes with spotted cell microarrays reveals mating-pheromone response genes}",
   Journal="Genome Biol.",
   Year="2006",
   Volume="7",
   Number="1",
   Pages="R6",
   Abstract={We have developed spotted cell microarrays for measuring cellular phenotypes on a large scale. Collections of cells are printed, stained for subcellular features, then imaged via automated, high-throughput microscopy, allowing systematic phenotypic characterization. We used this technology to identify genes involved in the response of yeast to mating pheromone. Besides morphology assays, cell microarrays should be valuable for high-throughput in situ hybridization and immunoassays, enabling new classes of genetic assays based on cell imaging.}
}

% 16495043 
@Article{pmid16495043,
   Author="Chu, T. C.  and Shieh, F.  and Lavery, L. A.  and Levy, M.  and Richards-Kortum, R.  and Korgel, B. A.  and Ellington, A. D. ",
   Title="{{L}abeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates}",
   Journal="Biosens Bioelectron",
   Year="2006",
   Volume="21",
   Number="10",
   Pages="1859--1866",
   Month="Apr",
   Abstract={Aptamers that bind to prostate specific membrane antigen (PSMA) were conjugated to luminescent CdSe and CdTe nanocrystals for cell-labeling studies. The aptamer-nanocrystal conjugates showed specific targeting of both fixed and live cells that overexpressed PSMA. More importantly, aptamers were able to label cells dispersed in a collagen gel matrix simulating tissue. The specific binding abilities and synthetic accessibility of aptamers combined with the photostability and small size of semiconductor nanocrystals offers a powerful and general tool for cellular imaging.}
}

% 16314457 
@Article{pmid16314457,
   Author="Bayer, T. S.  and Booth, L. N.  and Knudsen, S. M.  and Ellington, A. D. ",
   Title="{{A}rginine-rich motifs present multiple interfaces for specific binding by {R}{N}{A}}",
   Journal="RNA",
   Year="2005",
   Volume="11",
   Number="12",
   Pages="1848--1857",
   Month="Dec",
   Abstract={A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.}
}

% 16306980 
@Article{pmid16306980,
   Author="Levskaya, A.  and Chevalier, A. A.  and Tabor, J. J.  and Simpson, Z. B.  and Lavery, L. A.  and Levy, M.  and Davidson, E. A.  and Scouras, A.  and Ellington, A. D.  and Marcotte, E. M.  and Voigt, C. A. ",
   Title="{{S}ynthetic biology: engineering {E}scherichia coli to see light}",
   Journal="Nature",
   Year="2005",
   Volume="438",
   Number="7067",
   Pages="441--442",
   Month="Nov",
   Abstract={We have designed a bacterial system that is switched between different states by red light. The system consists of a synthetic sensor kinase that allows a lawn of bacteria to function as a biological film, such that the projection of a pattern of light on to the bacteria produces a high-definition (about 100 megapixels per square inch), two-dimensional chemical image. This spatial control of bacterial gene expression could be used to 'print' complex biological materials, for example, and to investigate signalling pathways through precise spatial and temporal control of their phosphorylation steps.}
}

% 16254932 
@Article{pmid16254932,
   Author="Levy, M.  and Cater, S. F.  and Ellington, A. D. ",
   Title="{{Q}uantum-dot aptamer beacons for the detection of proteins}",
   Journal="Chembiochem",
   Year="2005",
   Volume="6",
   Number="12",
   Pages="2163--2166",
   Month="Dec"
}

% 16246910 
@Article{pmid16246910,
   Author="Bryant, K. F.  and Cox, J. C.  and Wang, H.  and Hogle, J. M.  and Ellington, A. D.  and Coen, D. M. ",
   Title="{{B}inding of herpes simplex virus-1 {U}{S}11 to specific {R}{N}{A} sequences}",
   Journal="Nucleic Acids Res.",
   Year="2005",
   Volume="33",
   Number="19",
   Pages="6090--6100",
   Abstract={Herpes simplex virus-1 US11 is a RNA-binding protein with a novel RNA-binding domain. US11 has been reported to exhibit sequence- and conformation-specific RNA-binding, but the sequences and conformations important for binding are not known. US11 has also been described as a double-stranded RNA (dsRNA)-binding protein. To investigate the US11-RNA interaction, we performed in vitro selection of RNA aptamers that bind US11 from a RNA library consisting of >10(14) 80 base sequences which differ in a 30 base randomized region. US11 bound specifically to selected aptamers with an affinity of 70 nM. Analysis of 23 selected sequences revealed a strong consensus sequence. The US11 RNA-binding domain and < or =46 bases of selected RNA containing the consensus sequence were each sufficient for binding. US11 binding protected the consensus motif from hydroxyl radical cleavage. RNase digestions of a selected aptamer revealed regions of both single-stranded RNA and dsRNA. We observed that US11 bound two different dsRNAs in a sequence non-specific manner, but with lower affinity than it bound selected aptamers. The results define a relatively short specific sequence that binds US11 with high affinity and indicate that dsRNA alone does not confer high-affinity binding.}
}

% 16239467 
@Article{pmid16239467,
   Author="Ellington, A. D.  and Bull, J. J. ",
   Title="{{E}volution. {C}hanging the cofactor diet of an enzyme}",
   Journal="Science",
   Year="2005",
   Volume="310",
   Number="5747",
   Pages="454--455",
   Month="Oct"
}

% 16237122 
@Article{pmid16237122,
   Author="Pai, S.  and Ellington, A. D.  and Levy, M. ",
   Title="{{P}roximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores}",
   Journal="Nucleic Acids Res.",
   Year="2005",
   Volume="33",
   Number="18",
   Pages="e162",
   Abstract={The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.}
}

% 16217837 
@Article{pmid16217837,
   Author="Keller, K. M.  and Breeden, M. M.  and Zhang, J.  and Ellington, A. D.  and Brodbelt, J. S. ",
   Title="{{E}lectrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity}",
   Journal="J Mass Spectrom",
   Year="2005",
   Volume="40",
   Number="10",
   Pages="1327--1337",
   Month="Oct",
   Abstract={Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.}
}

% 16199170 
@Article{pmid16199170,
   Author="Collett, J. R.  and Cho, E. J.  and Ellington, A. D. ",
   Title="{{P}roduction and processing of aptamer microarrays}",
   Journal="Methods",
   Year="2005",
   Volume="37",
   Number="1",
   Pages="4--15",
   Month="Sep",
   Abstract={Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5' RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer microarrays, including blocking, washing, drying, and scanning. Examples are shown for the specific capture of fluorescently labeled target proteins either alone in binding buffer or in competition with labeled intracellular proteins from cell lysates. Consideration is given to the challenges involved in producing multiplex aptamer chips composed of aptamers taken from disparate literature sources, and to the development of standardized methods for characterizing the performance of capture reagents used in biosensors.}
}

% 16131588 
@Article{pmid16131588,
   Author="Levy, M.  and Griswold, K. E.  and Ellington, A. D. ",
   Title="{{D}irect selection of trans-acting ligase ribozymes by in vitro compartmentalization}",
   Journal="RNA",
   Year="2005",
   Volume="11",
   Number="10",
   Pages="1555--1562",
   Month="Oct",
   Abstract={We have used a compartmentalized in vitro selection method to directly select for ligase ribozymes that are capable of acting on and turning over separable oligonucleotide substrates. Starting from a degenerate pool, we selected a trans-acting variant of the Bartel class I ligase which statistically may have been the only active variant in the starting pool. The isolation of this sequence from the population suggests that this selection method is extremely robust at selecting optimal ribozymes and should, therefore, prove useful for the selection and optimization of other trans-acting nucleic acid catalysts capable of multiple turnover catalysis.}
}

% 16012175 
@Article{pmid16012175,
   Author="Flores, H.  and Ellington, A. D. ",
   Title="{{A} modified consensus approach to mutagenesis inverts the cofactor specificity of {B}acillus stearothermophilus lactate dehydrogenase}",
   Journal="Protein Eng. Des. Sel.",
   Year="2005",
   Volume="18",
   Number="8",
   Pages="369--377",
   Month="Aug",
   Abstract={Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD+. There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD+. A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN+. A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD+ than the wild-type enzyme and was weakly active with NADP+ and NMN+. An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP+ far better than the wild-type enzyme, it actually inverted its preference for NAD+ and NADP+.}
}

% 15769669 
@Article{pmid15769669,
   Author="Yan, A. C.  and Bell, K. M.  and Breeden, M. M.  and Ellington, A. D. ",
   Title="{{A}ptamers: prospects in therapeutics and biomedicine}",
   Journal="Front. Biosci.",
   Year="2005",
   Volume="10",
   Pages="1802--1827",
   Abstract={Most biopolymer drugs to date have been proteins. However, the ability to select nucleic acid binding species (aptamers) has led to the development of protein inhibitors and modulators that are small, readily synthesized nucleic acids. The techniques for optimizing, stabilizing, and delivering nucleic acid therapies are just beginning to be developed, but the same engineering flexibility that has so far allowed the generation of multiple, high affinity and specificity binding species appears to also apply to the methods for adapting nucleic acids to clinical applications. We review the selection and characterization of various aptamers and their applications to a variety of disease states, and then focus on the hurdles that must be overcome for the use of aptamers as both exogenously delivered drugs and as gene therapies.}
}

% 15734551 
@Article{pmid15734551,
   Author="Davidson, E. A.  and Ellington, A. D. ",
   Title="{{E}ngineering regulatory {R}{N}{A}s}",
   Journal="Trends Biotechnol.",
   Year="2005",
   Volume="23",
   Number="3",
   Pages="109--112",
   Month="Mar",
   Abstract={RNA has long been a favoured medium for in vitro evolution and engineering. Functional RNAs produced in vitro can bind small molecules (aptamers), possess catalytic activity (ribozymes) or do both (aptazymes). A plethora of recent work has shown similar strategies used naturally for gene regulation in bacteria. Interest in these natural systems has inspired an effort to engineer and evolve this activity in vivo. A recent paper by Isaacs et al. describes the engineering and in vivo activity of a small RNA that removes translation inhibition by binding the 5' untranslated region of its target mRNA and making the ribosome-binding site accessible.}
}

% 15713061 
@Article{pmid15713061,
   Author="Cho, E. J.  and Yang, L.  and Levy, M.  and Ellington, A. D. ",
   Title="{{U}sing a deoxyribozyme ligase and rolling circle amplification to detect a non-nucleic acid analyte, {A}{T}{P}}",
   Journal="J. Am. Chem. Soc.",
   Year="2005",
   Volume="127",
   Number="7",
   Pages="2022--2023",
   Month="Feb",
   Abstract={An allosteric ribozyme (aptazyme) has been used to transduce the binding of a small organic analyte (ATP) into the ligation of a circular template for rolling circle amplification (RCA). An ATP-activated deoxyribozyme ligase was immobilized on a glass slide and, upon addition of ATP, catalyzed the ligation of a circular padlock probe. The ligated products could be directly amplified and visualized via RCA. The coupled reaction exhibited could detect as little as 1 muM of ATP and could discriminate against structurally similar nucleotides such as GTP, CTP, and UTP. Cooperative ATP activation of the deoxyribozyme was faithfully mimicked by RCA, yielding an amplified "switch" that was responsive to ATP concentration.}
}

% 15707941 
@Article{pmid15707941,
   Author="Collett, J. R.  and Cho, E. J.  and Lee, J. F.  and Levy, M.  and Hood, A. J.  and Wan, C.  and Ellington, A. D. ",
   Title="{{F}unctional {R}{N}{A} microarrays for high-throughput screening of antiprotein aptamers}",
   Journal="Anal. Biochem.",
   Year="2005",
   Volume="338",
   Number="1",
   Pages="113--123",
   Month="Mar",
   Abstract={High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.}
}

% 15677335 
@Article{pmid15677335,
   Author="Hughes, R. A.  and Ellington, A. D. ",
   Title="{{M}istakes in translation don't translate into termination}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2005",
   Volume="102",
   Number="5",
   Pages="1273--1274",
   Month="Feb"
}

% 15600355 
@Article{pmid15600355,
   Author="Manimala, J. C.  and Wiskur, S. L.  and Ellington, A. D.  and Anslyn, E. V. ",
   Title="{{T}uning the specificity of a synthetic receptor using a selected nucleic acid receptor}",
   Journal="J. Am. Chem. Soc.",
   Year="2004",
   Volume="126",
   Number="50",
   Pages="16515--16519",
   Month="Dec",
   Abstract={Because of their relative simplicity, synthetic receptors often lack the selectivity observed for biopolymer receptors, such as aptamers. However, aptamer recognition of ligands is limited by the chemistries inherent in the four canonical nucleotides. Here, we report the design and selection of a ternary complex in which the specificity of a bis-boronic acid synthetic host (1) that binds to various carboxylic acids is tuned by a surrounding aptamer. Although, the synthetic receptor alone has higher selectivity for citrate over DL-tartrate, the formation of the aptamer:receptor complex reversed the organic host selectivity to preferentially bind tartrate. The RNA conformation changed upon the introduction of the synthetic host, consistent with an induced-fit mechanism for binding.}
}

% 15556407 
@Article{pmid15556407,
   Author="Hughes, R. A.  and Robertson, M. P.  and Ellington, A. D.  and Levy, M. ",
   Title="{{T}he importance of prebiotic chemistry in the {R}{N}{A} world}",
   Journal="Curr Opin Chem Biol",
   Year="2004",
   Volume="8",
   Number="6",
   Pages="629--633",
   Month="Dec",
   Abstract={In vitro selection experiments have clearly demonstrated that RNA can perform many of the functions necessary to support an RNA world. Moreover, it appears that novel functions could have readily evolved from existing functional RNA molecules. Therefore, diverse molecular ecosystems could potentially have arisen from an initial, small population of functional replicators. These findings suggest that the sequences of living systems may have been determined in part by chance occurrences at origins. Any extrapolations linking sequences (as opposed to functions) obtained in the laboratory to what may have occurred ca. 4 billion years ago are tenuous at best. Thus, perhaps the best way to understand origins is not by examining relatively unconstrained sequence information, but by examining the inherent constraints imposed by prebiotic chemistry.}
}

% 15461425 
@Article{pmid15461425,
   Author="Meyers, L. A.  and Lee, J. F.  and Cowperthwaite, M.  and Ellington, A. D. ",
   Title="{{T}he robustness of naturally and artificially selected nucleic acid secondary structures}",
   Journal="J. Mol. Evol.",
   Year="2004",
   Volume="58",
   Number="6",
   Pages="681--691",
   Month="Jun",
   Abstract={Thermodynamic stability and mutational robustness of secondary structure are critical to the function and evolutionary longevity of RNA molecules. We hypothesize that natural and artificial selection for functional molecules favors the formation of structures that are stable to both thermal and mutational perturbation. There is little direct evidence, however, that functional RNA molecules have been selected for their stability. Here we use thermodynamic secondary structure prediction algorithms to compare the thermal and mutational robustness of over 1000 naturally and artificially evolved molecules. Although we find evidence for the evolution of both types of stability in both sets of molecules, the naturally evolved functional RNA molecules were significantly more stable than those selected in vitro, and artificially evolved catalysts (ribozymes) were more stable than artificially evolved binding species (aptamers). The thermostability of RNA molecules bred in the laboratory is probably not constrained by a lack of suitable variation in the sequence pool but, rather, by intrinsic biases in the selection process.}
}

% 15300257 
@Article{pmid15300257,
   Author="Chelliserrykattil, J.  and Ellington, A. D. ",
   Title="{{E}volution of a {T}7 {R}{N}{A} polymerase variant that transcribes 2'-{O}-methyl {R}{N}{A}}",
   Journal="Nat. Biotechnol.",
   Year="2004",
   Volume="22",
   Number="9",
   Pages="1155--1160",
   Month="Sep",
   Abstract={Modified RNA and DNA molecules have novel properties that their natural counterparts do not possess, such as better resistance to degradation in cells and improved pharmacokinetic behavior. In particular, modifications at the 2'-OH of ribose are important for enhancing the stability of RNA. Unfortunately, it is difficult to enzymatically synthesize modified nucleic acids of any substantial length because natural polymerases incorporate modified nucleotides inefficiently. Previously, we reported an activity-based method for selecting functional T7 RNA polymerase variants based on the ability of a T7 RNA polymerase to reproduce itself. Here, we have modified the original procedure to identify polymerases that can efficiently incorporate multiple modified nucleotides at the 2' position of the ribose. Most important, our method allows the selection of polymerases that have good processivities and can be combined to simultaneously incorporate several different modified nucleotides in a transcript.}
}

% 15253644 
@Article{pmid15253644,
   Author="Kirby, R.  and Cho, E. J.  and Gehrke, B.  and Bayer, T.  and Park, Y. S.  and Neikirk, D. P.  and McDevitt, J. T.  and Ellington, A. D. ",
   Title="{{A}ptamer-based sensor arrays for the detection and quantitation of proteins}",
   Journal="Anal. Chem.",
   Year="2004",
   Volume="76",
   Number="14",
   Pages="4066--4075",
   Month="Jul",
   Abstract={Aptamer biosensors have been immobilized on beads, introduced into micromachined chips on the electronic tongue sensor array, and used for the detection and quantitation of proteins. Aptamer chips could detect proteins in both capture and sandwich assay formats. Unlike most protein-based arrays, the aptamer chips could be stripped and reused multiple times. The aptamer chips proved to be useful for screening aptamers from in vitro selection experiments and for sensitively quantitating the biothreat agent ricin.}
}

% 15167801 
@Article{pmid15167801,
   Author="Savran, C. A.  and Knudsen, S. M.  and Ellington, A. D.  and Manalis, S. R. ",
   Title="{{M}icromechanical detection of proteins using aptamer-based receptor molecules}",
   Journal="Anal. Chem.",
   Year="2004",
   Volume="76",
   Number="11",
   Pages="3194--3198",
   Month="Jun",
   Abstract={We report label-free protein detection using a microfabricated cantilever-based sensor that is functionalized with DNA aptamers to act as receptor molecules. The sensor utilizes two adjacent cantilevers that constitute a sensor/reference pair and allows direct detection of the differential bending between the two cantilevers. One cantilever is functionalized with aptamers selected for Taq DNA polymerase while the other is blocked with single-stranded DNA. We have found that the polymerase-aptamer binding induces a change in surface stress, which causes a differential cantilever bending that ranges from 3 to 32 nm depending on the ligand concentration. Protein recognition on the sensor surface is specific and has a concentration dependence that is similar to that in solution.}
}

% 15048102 
@Article{pmid15048102,
   Author="Knudsen, S. M.  and Ellington, A. D. ",
   Title="{{R}ibozyme déjà vu}",
   Journal="Nat. Struct. Mol. Biol.",
   Year="2004",
   Volume="11",
   Number="4",
   Pages="301--303",
   Month="Apr"
}

% 14681590 
@Article{pmid14681590,
   Author="Robertson, M. P.  and Knudsen, S. M.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of ribozymes dependent on peptides for activity}",
   Journal="RNA",
   Year="2004",
   Volume="10",
   Number="1",
   Pages="114--127",
   Month="Jan",
   Abstract={A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).}
}

% 14681388 
@Article{pmid14681388,
   Author="Hoffman, M. M.  and Khrapov, M. A.  and Cox, J. C.  and Yao, J.  and Tong, L.  and Ellington, A. D. ",
   Title="{{A}{A}{N}{T}: the {A}mino {A}cid-{N}ucleotide {I}nteraction {D}atabase}",
   Journal="Nucleic Acids Res.",
   Year="2004",
   Volume="32",
   Number="Database issue",
   Pages="D174--181",
   Month="Jan",
   Abstract={We have created an Amino Acid-Nucleotide Interaction Database (AANT; http://aant.icmb.utexas. edu/) that categorizes all amino acid-nucleotide interactions from experimentally determined protein-nucleic acid structures, and provides users with a graphic interface for visualizing these interactions in aggregate. AANT accomplishes this by extracting individual amino acid-nucleotide interactions from structures in the Protein Data Bank, combining and superimposing these interactions into multiple structure files (e.g. 20 amino acids x 5 nucleotides) and grouping structurally similar interactions into more readily identifiable clusters. Using the Chime web browser plug-in, users can view 3D representations of the superimpositions and clusters. The unique collection and representation of data on amino acid-nucleotide interactions facilitates understanding the specificity of protein-nucleic acid interactions at a more fundamental level, and allows comparison of otherwise extremely disparate sets of structures. Moreover, by modularly representing the fundamental interactions that govern binding specificity it may prove possible to better engineer nucleic acid binding proteins.}
}

% 14681367 
@Article{pmid14681367,
   Author="Lee, J. F.  and Hesselberth, J. R.  and Meyers, L. A.  and Ellington, A. D. ",
   Title="{{A}ptamer database}",
   Journal="Nucleic Acids Res.",
   Year="2004",
   Volume="32",
   Number="Database issue",
   Pages="95--100",
   Month="Jan",
   Abstract={The aptamer database is designed to contain comprehensive sequence information on aptamers and unnatural ribozymes that have been generated by in vitro selection methods. Such data are not normally collected in 'natural' sequence databases, such as GenBank. Besides serving as a storehouse of sequences that may have diagnostic or therapeutic utility, the database serves as a valuable resource for theoretical biologists who describe and explore fitness landscapes. The database is updated monthly and is publicly available at http://aptamer. icmb.utexas.edu/.}
}

% 14674448 
@Article{pmid14674448,
   Author="Ali, M. F.  and Kirby, R.  and Goodey, A. P.  and Rodriguez, M. D.  and Ellington, A. D.  and Neikirk, D. P.  and McDevitt, J. T. ",
   Title="{{D}{N}{A} hybridization and discrimination of single-nucleotide mismatches using chip-based microbead arrays}",
   Journal="Anal. Chem.",
   Year="2003",
   Volume="75",
   Number="18",
   Pages="4732--4739",
   Month="Sep",
   Abstract={The development of a chip-based sensor array composed of individually addressable agarose microbeads has been demonstrated for the rapid detection of DNA oligonucleotides. Here, a "plug and play" approach allows for the simple incorporation of various biotinylated DNA capture probes into the bead-microreactors, which are derivatized in each case with avidin docking sites. The DNA capture probe containing microbeads are selectively arranged in micromachined cavities localized on silicon wafers. The microcavities possess trans-wafer openings, which allow for both fluid flow through the microreactors/analysis chambers and optical access to the chemically sensitive microbeads. Collectively, these features allow the identification and quantitation of target DNA analytes to occur in near real time using fluorescence changes that accompany binding of the target sample. The unique three-dimensional microenvironment within the agarose bead and the microfluidics capabilities of the chip structure afford a fully integrated package that fosters rapid analyses of solutions containing complex mixtures of DNA oligomers. These analyses can be completed at room temperature through the use of appropriate hybridization buffers. For applications requiring analysis of < or = 10(2) different DNA sequences, the hybridization times and point mutation selectivity factors exhibited by this bead array method exceed in many respects the operational characteristics of the commonly utilized planar DNA chip technologies. The power and utility of this microbead array DNA detection methodology is demonstrated here for the analysis of fluids containing a variety of similar 18-base oligonucleotides. Hybridization times on the order of minutes with point mutation selectivity factors greater than 10000 and limit of detection values of approximately 10(-13) M are obtained readily with this microbead array system.}
}

% 14667253 
@Article{pmid14667253,
   Author="Bacher, J. M.  and Bull, J. J.  and Ellington, A. D. ",
   Title="{{E}volution of phage with chemically ambiguous proteomes}",
   Journal="BMC Evol. Biol.",
   Year="2003",
   Volume="3",
   Pages="24",
   Month="Dec",
   Abstract={The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.\\ The bacteriophage Qbeta initially grows poorly in the presence of the amino acid analogue 6-fluorotryptophan. After 25 serial passages, the fitness of the phage on the analogue was substantially increased; there was no loss of fitness when the evolved phage were passaged in the presence of tryptophan. Seven mutations were fixed throughout the phage in two independent lines of descent. None of the mutations changed a tryptophan residue.\\ A relatively small number of mutations allowed an unnatural amino acid to be functionally incorporated into a highly interdependent set of proteins. These results support the 'ambiguous intermediate' hypothesis for the emergence of divergent genetic codes, in which the adoption of a new genetic code is preceded by the evolution of proteins that can simultaneously accommodate more than one amino acid at a given codon. It may now be possible to direct the evolution of organisms with novel genetic codes using methods that promote ambiguous intermediates.}
}

% 14500834 
@Article{pmid14500834,
   Author="Rajendran, M.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of molecular beacons}",
   Journal="Nucleic Acids Res.",
   Year="2003",
   Volume="31",
   Number="19",
   Pages="5700--5713",
   Month="Oct",
   Abstract={While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.}
}

% 12949563 
@Article{pmid12949563,
   Author="Tabor, J. J.  and Ellington, A. D. ",
   Title="{{P}laying to win at {D}{N}{A} computation}",
   Journal="Nat. Biotechnol.",
   Year="2003",
   Volume="21",
   Number="9",
   Pages="1013--1015",
   Month="Sep"
}

% 12824567 
@Article{pmid12824567,
   Author="Chelliserrykattil, J.  and Ellington, A. D. ",
   Title="{{A}utogene selections}",
   Journal="Methods Mol. Biol.",
   Year="2003",
   Volume="230",
   Pages="27--43"
}

% 12743371 
@Article{pmid12743371,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{E}xponential growth by cross-catalytic cleavage of deoxyribozymogens}",
   Journal="Proc. Natl. Acad. Sci. U.S.A.",
   Year="2003",
   Volume="100",
   Number="11",
   Pages="6416--6421",
   Month="May",
   Abstract={We have designed an autocatalytic cycle based on the highly efficient 10-23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10-23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an in vitro selection experiment conducted in the absence of any protein enzymes.}
}

% 12698297 
@Article{pmid12698297,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{P}eptide-templated nucleic acid ligation}",
   Journal="J. Mol. Evol.",
   Year="2003",
   Volume="56",
   Number="5",
   Pages="607--615",
   Month="May",
   Abstract={Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated with a 5'-iodine and a 3'phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that peptide and nucleic acid replicators could have been interdependent.}
}

% 12531194 
@Article{pmid12531194,
   Author="Hesselberth, J. R.  and Robertson, M. P.  and Knudsen, S. M.  and Ellington, A. D. ",
   Title="{{S}imultaneous detection of diverse analytes with an aptazyme ligase array}",
   Journal="Anal. Biochem.",
   Year="2003",
   Volume="312",
   Number="2",
   Pages="106--112",
   Month="Jan",
   Abstract={Allosteric ribozymes (aptazymes) can transduce the noncovalent recognition of analytes into the catalytic generation of readily observable signals. Aptazymes are easily engineered, can detect diverse classes of biologically relevant molecules, and have high signal-to-noise ratios. These features make aptazymes useful candidates for incorporation into biosensor arrays. Allosteric ribozyme ligases that can recognize a variety of analytes ranging from small organics to proteins have been generated. Upon incorporation into an array format, multiple different aptazyme ligases were able to simultaneously detect their cognate analytes with high specificity. Analyte concentrations could be accurately measured into the nanomolar range. The fact that analytes induced the formation of new covalent bonds in aptazyme ligases (as opposed to noncovalent bonds in antibodies) potentiated stringent washing of the array, leading to improved signal-to-noise ratios and limits of detection.}
}

% 12466564 
@Article{pmid12466564,
   Author="Yang, X.  and Bassett, S. E.  and Li, X.  and Luxon, B. A.  and Herzog, N. K.  and Shope, R. E.  and Aronson, J.  and Prow, T. W.  and Leary, J. F.  and Kirby, R.  and Ellington, A. D.  and Gorenstein, D. G. ",
   Title="{{C}onstruction and selection of bead-bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid {P}{C}{R}-based sequencing}",
   Journal="Nucleic Acids Res.",
   Year="2002",
   Volume="30",
   Number="23",
   Pages="e132",
   Month="Dec",
   Abstract={Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.}
}

% 12466025 
@Article{pmid12466025,
   Author="Thompson, K. M.  and Syrett, H. A.  and Knudsen, S. M.  and Ellington, A. D. ",
   Title="{{G}roup {I} aptazymes as genetic regulatory switches}",
   Journal="BMC Biotechnol.",
   Year="2002",
   Volume="2",
   Pages="21",
   Month="Dec",
   Abstract={Allosteric ribozymes (aptazymes) that have extraordinary activation parameters have been generated in vitro by design and selection. For example, hammerhead and ligase ribozymes that are activated by small organic effectors and protein effectors have been selected from random sequence pools appended to extant ribozymes. Many ribozymes, especially self-splicing introns, are known control gene regulation or viral replication in vivo. We attempted to generate Group I self-splicing introns that were activated by a small organic effector, theophylline, and to show that such Group I aptazymes could mediate theophylline-dependent splicing in vivo.\\ By appending aptamers to the Group I self-splicing intron, we have generated a Group I aptazyme whose in vivo splicing is controlled by exogenously added small molecules. Substantial differences in gene regulation could be observed with compounds that differed by as little as a single methyl group. The effector-specificity of the Group I aptazyme could be rationally engineered for new effector molecules.\\ Group I aptazymes may find applications as genetic regulatory switches for generating conditional knockouts at the level of mRNA or for developing economically viable gene therapies.}
}

% 12447352 
@Article{pmid12447352,
   Author="Hesselberth, J. R.  and Ellington, A. D. ",
   Title="{{A} (ribo) switch in the paradigms of genetic regulation}",
   Journal="Nat. Struct. Biol.",
   Year="2002",
   Volume="9",
   Number="12",
   Pages="891--893",
   Month="Dec"
}

% 12384610 
@Article{pmid12384610,
   Author="Cox, J. C.  and Hayhurst, A.  and Hesselberth, J.  and Bayer, T. S.  and Georgiou, G.  and Ellington, A. D. ",
   Title="{{A}utomated selection of aptamers against protein targets translated in vitro: from gene to aptamer}",
   Journal="Nucleic Acids Res.",
   Year="2002",
   Volume="30",
   Number="20",
   Pages="e108",
   Month="Oct",
   Abstract={Reagents for proteome research must of necessity be generated by high throughput methods. Aptamers are potentially useful as reagents to identify and quantitate individual proteins, yet are currently produced for the most part by manual selection procedures. We have developed automated selection methods, but must still individually purify protein targets. Therefore, we have attempted to select aptamers against protein targets generated by in vitro transcription and translation of individual genes. In order to specifically immobilize the protein targets for selection, they are also biotinylated in vitro. As a proof of this method, we have selected aptamers against translated human U1A, a component of the nuclear spliceosome. Selected sequences demonstrated exquisite mimicry of natural binding sequences and structures. These results not only reveal a potential path to the high throughput generation of aptamers, but also yield insights into the incredible specificity of the U1A protein for its natural RNA ligands.}
}

% 12204684 
@Article{pmid12204684,
   Author="Sooter, L. J.  and Ellington, A. D. ",
   Title="{{R}eflections on a novel therapeutic candidate}",
   Journal="Chem. Biol.",
   Year="2002",
   Volume="9",
   Number="8",
   Pages="857--858",
   Month="Aug",
   Abstract={Aptamers composed of L-nucleic acids, Spiegelmers, were selected to specifically bind GnRH. Spiegelmer inhibition of GnRH activity was demonstrated in both cellular and animal models. Rabbit studies showed minimal immunogenic response to the agents.}
}

% 12186650 
@Article{pmid12186650,
   Author="Bacher, J. M.  and Reiss, B. D.  and Ellington, A. D. ",
   Title="{{A}nticipatory evolution and {D}{N}{A} shuffling}",
   Journal="Genome Biol.",
   Year="2002",
   Volume="3",
   Number="8",
   Pages="REVIEWS1021",
   Month="Jul",
   Abstract={DNA shuffling has proven to be a powerful technique for the directed evolution of proteins. A mix of theoretical and applied research has now provided insights into how recombination can be guided to more efficiently generate proteins and even organisms with altered functions.}
}

% 12165173 
@Article{pmid12165173,
   Author="Yan, A.  and Ellington, A. D. ",
   Title="{{A}ptamers as potential diagnostic reagents for diabetes}",
   Journal="Diabetes Technol. Ther.",
   Year="2002",
   Volume="4",
   Number="3",
   Pages="339--346"
}

% 12089558 
@Article{pmid12089558,
   Author="Hartig, J. S.  and Najafi-Shoushtari, S. H.  and Grune, I.  and Yan, A.  and Ellington, A. D.  and Famulok, M. ",
   Title="{{P}rotein-dependent ribozymes report molecular interactions in real time}",
   Journal="Nat. Biotechnol.",
   Year="2002",
   Volume="20",
   Number="7",
   Pages="717--722",
   Month="Jul",
   Abstract={Most approaches to monitoring interactions between biological macromolecules require large amounts of material, rely upon the covalent modification of an interaction partner, or are not amenable to real-time detection. We have developed a generalizable assay system based on interactions between proteins and reporter ribozymes. The assay can be configured in a modular fashion to monitor the presence and concentration of a protein or of molecules that modulate protein function. We report two applications of the assay: screening for a small molecule that disrupts protein binding to its nucleic acid target and screening for protein protein interactions. We screened a structurally diverse library of antibiotics for small molecules that modulate the activity of HIV-1 Rev-responsive ribozymes by binding to Rev. We identified an inhibitor that subsequently inhibited HIV-1 replication in cells. A simple format switch allowed reliable monitoring of domain-specific interactions between the blood-clotting factor thrombin and its protein partners. The rapid identification of interactions between proteins or of compounds that disrupt such interactions should have substantial utility for the drug-discovery process.}
}

% 12052180 
@Article{pmid12052180,
   Author="Cox, J. C.  and Rajendran, M.  and Riedel, T.  and Davidson, E. A.  and Sooter, L. J.  and Bayer, T. S.  and Schmitz-Brown, M.  and Ellington, A. D. ",
   Title="{{A}utomated acquisition of aptamer sequences}",
   Journal="Comb. Chem. High Throughput Screen.",
   Year="2002",
   Volume="5",
   Number="4",
   Pages="289--299",
   Month="Jun",
   Abstract={While the in vitro selection of nucleic acid binding species (aptamers) requires numerous liquid-handling steps, these steps are relatively straightforward and the overall process is therefore amenable to automation. Here we demonstrate that automated selection techniques are capable of generating aptamers against a number of diverse protein targets. Automated selection techniques can be integrated with automated analytical methods, including sequencing, determination of binding constants, and structural analysis. The methods that have so far been developed can be further multiplexed, and it should soon be possible to attempt the selection of aptamers against organismal proteomes or metabolomes.}
}

% 12052178 
@Article{pmid12052178,
   Author="Rajendran, M.  and Ellington, A. D. ",
   Title="{{S}electing nucleic acids for biosensor applications}",
   Journal="Comb. Chem. High Throughput Screen.",
   Year="2002",
   Volume="5",
   Number="4",
   Pages="263--270",
   Month="Jun",
   Abstract={In vitro selection can be used to generate nucleic acid binding species (aptamers) and catalysts (ribozymes) that can recognize a variety of molecules. Because nucleic acid function is largely derived from readily tabulated secondary structures, it has proven possible to engineer aptamers and ribozymes to function as biosensors. Labeling nucleic acids with reporter molecules has yielded simple antibody substitutes, but by relying on ligand-dependent conformational changes it has also proven possible to generate biosensors that can recognize and specifically report the presence of ligands in homogenous solution. It may prove possible to generate signaling aptamers and allosteric ribozymes (aptazymes) that are responsive to a large fraction of an organismal proteome or metabolome using automated methods. Nucleic acid biosensor arrays for non-nucleic acid targets could likely be generated with the same facility as DNA chips.}
}

% 11983331 
@Article{pmid11983331,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{A}{T}{P}-dependent allosteric {D}{N}{A} enzymes}",
   Journal="Chem. Biol.",
   Year="2002",
   Volume="9",
   Number="4",
   Pages="417--426",
   Month="Apr",
   Abstract={Effector-activated ribozymes that respond to small organic molecules have previously been generated by appending binding species (aptamers) to ribozymes. In order to determine if deoxyribozymes can similarly be activated by effector molecules, we have appended an anti-adenosine aptamer to a selected deoxyribozyme ligase. The resultant constructs are specifically activated by ATP. Optimization of the joining region resulted in ligases that are activated up to 460-fold by ATP. The selected deoxyribozyme catalyzes ligation largely via a templating mechanism. Effector activation is surprisingly achieved by suppression of the rate of the background, templated ligation reaction in the absence of the effector molecule, probably by misalignment of the oligonucleotide substrates. This novel allosteric mechanism has not previously been observed for nucleic-acid catalysts and is rare even in protein catalysts.}
}

% 11821911 
@Article{pmid11821911,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of a deoxyribozyme that can utilize multiple substrates}",
   Journal="J. Mol. Evol.",
   Year="2002",
   Volume="54",
   Number="2",
   Pages="180--190",
   Month="Feb",
   Abstract={Deoxyribozymes that could catalyze the formation of an internucleotide phosphorothioester linkage were selected from a random sequence pool. During the course of the selection, the pool was successively challenged with five oligonucleotide substrates, each of which terminated in the same hexanucleotide sequence. Selected deoxyribozyme ligases could use all five substrates, albeit to different degrees, and appeared to form secondary structures that allow differential pairing between the deoxyribozyme and each substrate. These results suggest that early replicases may have been able to bind a variety of oligonucleotide substrates while catalyzing ligation via a common junction.}
}

% 11806761 
@Article{pmid11806761,
   Author="Chelliserrykattil, J.  and Cai, G.  and Ellington, A. D. ",
   Title="{{A} combined in vitro/in vivo selection for polymerases with novel promoter specificities}",
   Journal="BMC Biotechnol.",
   Year="2001",
   Volume="1",
   Pages="13",
   Abstract={The DNA-dependent RNA polymerase from T7 bacteriophage (T7 RNAP) has been extensively characterized, and like other phage RNA polymerases it is highly specific for its promoter. A combined in vitro/in vivo selection method has been developed for the evolution of T7 RNA polymerases with altered promoter specificities. Large (10(3)-10(6)) polymerase libraries were made and cloned downstream of variant promoters. Those polymerase variants that can recognize variant promoters self-amplify both themselves and their attendant mRNAs in vivo. Following RT / PCR amplification in vitro, the most numerous polymerase genes are preferentially cloned and carried into subsequent rounds of selection.\\ A T7 RNA polymerase library that was randomized at three positions was cloned adjacent to a T3-like promoter sequence, and a 'specialist' T7 RNA polymerase was identified. A library that was randomized at a different set of positions was cloned adjacent to a promoter library in which four positions had been randomized, and 'generalist' polymerases that could utilize a variety of T7 promoters were identified, including at least one polymerase with an apparently novel promoter specificity. This method may have applications for evolving other polymerase variants with novel phenotypes, such as the ability to incorporate modified nucleotides.}
}

% 11786015 
@Article{pmid11786015,
   Author="Flores, H.  and Ellington, A. D. ",
   Title="{{I}ncreasing the thermal stability of an oligomeric protein, beta-glucuronidase}",
   Journal="J. Mol. Biol.",
   Year="2002",
   Volume="315",
   Number="3",
   Pages="325--337",
   Month="Jan",
   Abstract={The reporter enzyme beta-glucuronidase was mutagenized and evolved for thermostability. After four cycles of screening the best variant was more active than the wild-type enzyme, and retained function at 70 degrees C, whereas the wild-type enzyme lost function at 65 degrees C. Variants derived from sequential mutagenesis were shuffled together, and re-screened for thermostability. The best variants retained activities at even higher temperatures (80 degrees C), but had specific activities that were now less than that of the wild-type enzyme. The mutations clustered near the tetramer interface of the enzyme, and many of the evolved variants showed much greater resistance to quaternary structure disruption at high temperatures, which is also a characteristic of naturally thermostable enzymes. Together, these results suggest a pathway for the evolution of thermostability in which enzymes initially become stable at high temperatures without loss of activity at low temperatures, while further evolution leads to enzymes that have kinetic parameters that are optimized for high temperatures.}
}

% 11768971 
@Article{pmid11768971,
   Author="Matsumura, I.  and Ellington, A. D. ",
   Title="{{M}utagenic polymerase chain reaction of protein-coding genes for in vitro evolution}",
   Journal="Methods Mol. Biol.",
   Year="2002",
   Volume="182",
   Pages="259--267"
}

% 11688716 
@Article{pmid11688716,
   Author="Sooter, L. J.  and Riedel, T.  and Davidson, E. A.  and Levy, M.  and Cox, J. C.  and Ellington, A. D. ",
   Title="{{T}oward automated nucleic acid enzyme selection}",
   Journal="Biol. Chem.",
   Year="2001",
   Volume="382",
   Number="9",
   Pages="1327--1334",
   Month="Sep",
   Abstract={Methods for automation of nucleic acid selections are being developed. The selection of aptamers has been successfully automated using a Biomek 2000 workstation. Several binding species with nanomolar affinities were isolated from diverse populations. Automation of a deoxyribozyme ligase selection is in progress. The process requires eleven times more robotic manipulations than an aptamer selection. The random sequence pool contained a 5' iodine residue and the ligation substrate contained a 3' phosphorothioate. Initially, a manual deoxyribozyme ligase selection was performed. Thirteen rounds of selection yielded ligators with a 400-fold increase in activity over the initial pool. Several difficulties were encountered during the automation of DNA catalyst selection, including effectively washing bead-bound DNA, pipetting 50% glycerol solutions, purifying single strand DNA, and monitoring the progress of the selection as it is performed. Nonetheless, automated selection experiments for deoxyribozyme ligases were carried out starting from either a naive pool or round eight of the manually selected pool. In both instances, the first round of selection revealed an increase in ligase activity. However, this activity was lost in subsequent rounds. A possible cause could be mispriming during the unmonitored PCR reactions. Potential solutions include pool redesign, fewer PCR cycles, and integration of a fluorescence microtiter plate reader to allow robotic 'observation' of the selections as they progress.}
}

% 11557346 
@Article{pmid11557346,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{S}election of deoxyribozyme ligases that catalyze the formation of an unnatural internucleotide linkage}",
   Journal="Bioorg. Med. Chem.",
   Year="2001",
   Volume="9",
   Number="10",
   Pages="2581--2587",
   Month="Oct",
   Abstract={The chemical ligation of DNA molecules can be mediated by terminal phosphorothioate displacement of a 5' iodine. We have selected deoxyribozymes that can catalyze the formation of such phosphorothioester internucleotide linkages. The selected deoxyribozymes enhance the rate of ligation in part through the provision of a template that aligns the ligation junction and do not appear to require metal ions for catalysis.}
}

% 11557339 
@Article{pmid11557339,
   Author="Cox, J. C.  and Ellington, A. D. ",
   Title="{{A}utomated selection of anti-protein aptamers}",
   Journal="Bioorg. Med. Chem.",
   Year="2001",
   Volume="9",
   Number="10",
   Pages="2525--2531",
   Month="Oct",
   Abstract={The in vitro selection of nucleic acid binding species (aptamers) is frequently repetitive, time-consuming, and poorly adapted to high-throughput applications. We have adapted automated workstations to select anti-protein aptamers; as an example, we demonstrated the selection of anti-lysozyme aptamers that function as efficient inhibitors of cell lysis. The increases in throughput brought about by automation should potentiate the application of aptamer technology to the rapidly growing field of proteomics.}
}

% 11525762 
@Article{pmid11525762,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{R}{N}{A} world: catalysis abets binding, but not vice versa}",
   Journal="Curr. Biol.",
   Year="2001",
   Volume="11",
   Number="16",
   Pages="R665--667",
   Month="Aug",
   Abstract={The recent selection of a complex ribozyme capable of general polymerization on a template in trans has revealed how catalysts may have arisen from one another in the RNA world.}
}

% 11514527 
@Article{pmid11514527,
   Author="Bacher, J. M.  and Ellington, A. D. ",
   Title="{{S}election and characterization of {E}scherichia coli variants capable of growth on an otherwise toxic tryptophan analogue}",
   Journal="J. Bacteriol.",
   Year="2001",
   Volume="183",
   Number="18",
   Pages="5414--5425",
   Month="Sep",
   Abstract={Escherichia coli isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (aroP) and tryptophanyl-tRNA synthetase (trpS) contained several amino acid substitutions, and the tyrosine repressor (tyrR) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.}
}

% 11433277 
@Article{pmid11433277,
   Author="Robertson, M. P.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of nucleoprotein enzymes}",
   Journal="Nat. Biotechnol.",
   Year="2001",
   Volume="19",
   Number="7",
   Pages="650--655",
   Month="Jul",
   Abstract={Natural nucleic acids frequently rely on proteins for stabilization or catalytic activity. In contrast, nucleic acids selected in vitro can catalyze a wide range of reactions even in the absence of proteins. To augment selected nucleic acids with protein functionalities, we have developed a technique for the selection of protein-dependent ribozyme ligases. After randomizing a previously selected ribozyme ligase, L1, we selected variants that required one of two protein cofactors, a tyrosyl transfer RNA (tRNA) synthetase (Cyt18) or hen egg white lysozyme. The resulting nucleoprotein enzymes were activated several thousand fold by their cognate protein effectors, and could specifically recognize the structures of the native proteins. Protein-dependent ribozymes can potentially be adapted to novel assays for detecting target proteins, and the selection method's generality may allow the high-throughput identification of ribozymes capable of recognizing a sizable fraction of a proteome.}
}

% 11427883 
@Article{pmid11427883,
   Author="Levy, M.  and Ellington, A. D. ",
   Title="{{T}he descent of polymerization}",
   Journal="Nat. Struct. Biol.",
   Year="2001",
   Volume="8",
   Number="7",
   Pages="580--582",
   Month="Jul"
}

% 11401165 
@Article{pmid11401165,
   Author="Christian, N. P.  and Reilly, J. P.  and Mokler, V. R.  and Wincott, F. E.  and Ellington, A. D. ",
   Title="{{E}lucidation of the initial step of oligonucleotide fragmentation in matrix-assisted laser desorption/ionization using modified nucleic acids}",
   Journal="J. Am. Soc. Mass Spectrom.",
   Year="2001",
   Volume="12",
   Number="6",
   Pages="744--753",
   Month="Jun",
   Abstract={To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.}
}

% 11369241 
@Article{pmid11369241,
   Author="Cox, J. C.  and Ellington, A. D. ",
   Title="{{D}{N}{A} computation function}",
   Journal="Curr. Biol.",
   Year="2001",
   Volume="11",
   Number="9",
   Pages="R336",
   Month="May"
}

% 11345430 
@Article{pmid11345430,
   Author="Robertson, M. P.  and Hesselberth, J. R.  and Ellington, A. D. ",
   Title="{{O}ptimization and optimality of a short ribozyme ligase that joins non-{W}atson-{C}rick base pairings}",
   Journal="RNA",
   Year="2001",
   Volume="7",
   Number="4",
   Pages="513--523",
   Month="Apr",
   Abstract={A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.}
}

% 11283579 
@Article{pmid11283579,
   Author="Hoffman, D.  and Hesselberth, J.  and Ellington, A. D. ",
   Title="{{S}witching nucleic acids for antibodies}",
   Journal="Nat. Biotechnol.",
   Year="2001",
   Volume="19",
   Number="4",
   Pages="313--314",
   Month="Apr"
}

% 11252780 
@Article{pmid11252780,
   Author="Matsumura, I.  and Olsen, M. J.  and Ellington, A. D. ",
   Title="{{O}ptimization of heterologous gene expression for in vitro evolution}",
   Journal="BioTechniques",
   Year="2001",
   Volume="30",
   Number="3",
   Pages="474--476",
   Month="Mar"
}

% 11124909 
@Article{pmid11124909,
   Author="Matsumura, I.  and Ellington, A. D. ",
   Title="{{I}n vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates}",
   Journal="J. Mol. Biol.",
   Year="2001",
   Volume="305",
   Number="2",
   Pages="331--339",
   Month="Jan",
   Abstract={The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K(m)) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wild-type and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity.}
}

% 11101810 
@Article{pmid11101810,
   Author="Jhaveri, S.  and Rajendran, M.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of signaling aptamers}",
   Journal="Nat. Biotechnol.",
   Year="2000",
   Volume="18",
   Number="12",
   Pages="1293--1297",
   Month="Dec",
   Abstract={Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 microM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general "switch" for transducing molecular recognition to optical signals.}
}

% 10943569 
@Article{pmid10943569,
   Author="Hesselberth, J.  and Robertson, M. P.  and Jhaveri, S.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of nucleic acids for diagnostic applications}",
   Journal="J. Biotechnol.",
   Year="2000",
   Volume="74",
   Number="1",
   Pages="15--25",
   Month="Mar",
   Abstract={In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as 'universal receptors' that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes.}
}

% 10889989 
@Article{pmid10889989,
   Author="Marshall, K. A.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} aptamers}",
   Journal="Meth. Enzymol.",
   Year="2000",
   Volume="318",
   Pages="193--214"
}

% 10786840 
@Article{pmid10786840,
   Author="Ellington, A. D.  and Khrapov, M.  and Shaw, C. A. ",
   Title="{{T}he scene of a frozen accident}",
   Journal="RNA",
   Year="2000",
   Volume="6",
   Number="4",
   Pages="485--498",
   Month="Apr",
   Abstract={It has been suggested that in vitro selection experiments can provide information not only on what might have occurred during the evolution of the RNA world, but can in fact yield insights into particular features of the RNA world. In particular, it has been suggested that the sequences of anti-amino acid aptamers can provide clues to the origin of the genetic code, and that there is a statistically significant association between motifs found in aptamers and codons. We argue that the suggested connections between modern motifs and ancient sequences are logically tenuous, and show that there is no statistically meaningful association between motifs found in aptamers and codons.}
}

% 10734194 
@Article{pmid10734194,
   Author="Robertson, M. P.  and Ellington, A. D. ",
   Title="{{D}esign and optimization of effector-activated ribozyme ligases}",
   Journal="Nucleic Acids Res.",
   Year="2000",
   Volume="28",
   Number="8",
   Pages="1751--1759",
   Month="Apr",
   Abstract={A selected ribozyme ligase, L1, has been engineered to respond to small organic effectors. Residues important for ribozyme catalysis were mapped to a compact core structure. Aptamers that bound adenosine and theophylline were appended to the core structure, and the resultant aptazymes proved to be responsive to their cognate effectors. Rational sequence substitutions in the joining region between the aptamer and the ribozyme yielded aptazymes whose activities were enhanced from 800-1600-fold in the presence of 1 mM ATP or theophylline, respectively. However, when an anti-flavin aptamer was appended to the core ribozyme structure flavin-responsivity was minimal. The joining region between the aptamer and the ribozyme core was randomized and a series of negative and positive selection steps yielded aptazymes that were activated by up to 260-fold in the presence of 100 microM FMN. The selected joining regions proved to be 'communication modules' that could be used to join other aptamers to the ribozyme core to form aptazymes. These results show that ribozyme ligases can be readily engineered to function as allosteric enzymes, and reveal that many of the techniques and principles previously demonstrated during the development of hammerhead aptazymes may be generalizable.}
}

% 10671532 
@Article{pmid10671532,
   Author="Hirao, I.  and Madin, K.  and Endo, Y.  and Yokoyama, S.  and Ellington, A. D. ",
   Title="{{R}{N}{A} aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin}",
   Journal="J. Biol. Chem.",
   Year="2000",
   Volume="275",
   Number="7",
   Pages="4943--4948",
   Month="Feb",
   Abstract={Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.}
}

% 10671531 
@Article{pmid10671531,
   Author="Hesselberth, J. R.  and Miller, D.  and Robertus, J.  and Ellington, A. D. ",
   Title="{{I}n vitro selection of {R}{N}{A} molecules that inhibit the activity of ricin {A}-chain}",
   Journal="J. Biol. Chem.",
   Year="2000",
   Volume="275",
   Number="7",
   Pages="4937--4942",
   Month="Feb",
   Abstract={The cytotoxin ricin disables translation by depurinating a conserved site in eukaryotic rRNA. In vitro selection has been used to generate RNA ligands (aptamers) specific for the catalytic ricin A-chain (RTA). The anti-RTA aptamers bear no resemblance to the normal RTA substrate, the sarcin-ricin loop (SRL), and were not depurinated by RTA. An initial 80-nucleotide RNA ligand was minimized to a 31-nucleotide aptamer that contained all sequences and structures necessary for interacting with RTA. This minimal RNA formed high affinity complexes with RTA (K(d) = 7.3 nM) which could compete directly with the SRL for binding to RTA. The aptamer inhibited RTA depurination of the SRL and could partially protect translation from RTA inhibition. The IC(50) of the aptamer for RTA in an in vitro translation assay is 100 nM, roughly 3 orders of magnitude lower than a small molecule inhibitor of ricin, pteroic acid, and 2 orders of magnitude lower than the best known RNA inhibitor. The novel anti-RTA aptamers may find application as diagnostic reagents for a potential biological warfare agent and hold promise as scaffolds for the development of strong ricin inhibitors.}
}

% 10542083 
@Article{pmid10542083,
   Author="Marshall, K. A.  and Ellington, A. D. ",
   Title="{{T}raining ribozymes to switch}",
   Journal="Nat. Struct. Biol.",
   Year="1999",
   Volume="6",
   Number="11",
   Pages="992--994",
   Month="Nov",
   Abstract={Ribozymes that are sensitive to cyclic nucleotides have been selected in vitro. Remarkably, the cGMP-dependent ribozymes are specifically activated by a factor of 5,000 - the largest allosteric ribozyme activation by a small molecule seen to date.}
}

% 10552046 
@Article{pmid10552046,
   Author="Marshall, K. A.  and Ellington, A. D. ",
   Title="{{M}olecular parasites that evolve longer genomes}",
   Journal="J. Mol. Evol.",
   Year="1999",
   Volume="49",
   Number="5",
   Pages="656--663",
   Month="Nov",
   Abstract={Molecular parasites that utilize the replication machinery of cells or of in vitro amplification reactions have previously been characterized. By and large, these parasites have been smaller than the viruses or amplicons that gave rise to them. This is likely because shorter genomes can be replicated more quickly. In contrast, we have identified and characterized parasites of an isothermal amplification reaction that are longer than their parental molecules yet replicate much more efficiently. These results raise interesting questions regarding whether the optimal size of replicators reflects a trade-off between the information encoded in a parasite and the information encoded in the machinery replicating that parasite.}
}

% 10472627 
@Article{pmid10472627,
   Author="James, K. D.  and Ellington, A. D. ",
   Title="{{T}he fidelity of template-directed oligonucleotide ligation and the inevitability of polymerase function}",
   Journal="Orig Life Evol Biosph",
   Year="1999",
   Volume="29",
   Number="4",
   Pages="375--390",
   Month="Aug",
   Abstract={The first living systems may have employed template-directed oligonucleotide ligation for replication. The utility of oligonucleotide ligation as a mechanism for the origin and evolution of life is in part dependent on its fidelity. We have devised a method for evaluating ligation fidelity in which ligation substrates are selected from random sequence libraries. The fidelities of chemical and enzymatic ligation are compared under a variety of conditions. While reaction conditions can be found that promote high fidelity copying, departure from these conditions leads to error-prone copying. In particular, ligation reactions with shorter oligonucleotide substrates are less efficient but more faithful. These results support a model for origins in which there was selective pressure for template-directed oligonucleotide ligation to be gradually supplanted by mononucleotide polymerization.}
}

% 10433691 
@Article{pmid10433691,
   Author="Zidek, L.  and Stone, M. J.  and Lato, S. M.  and Pagel, M. D.  and Miao, Z.  and Ellington, A. D.  and Novotny, M. V. ",
   Title="{{N}{M}{R} mapping of the recombinant mouse major urinary protein {I} binding site occupied by the pheromone 2-sec-butyl-4,5-dihydrothiazole}",
   Journal="Biochemistry",
   Year="1999",
   Volume="38",
   Number="31",
   Pages="9850--9861",
   Month="Aug",
   Abstract={The interactions between the mouse major urinary protein isoform MUP-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole have been characterized in solution. (15)N-labeled and (15)N, (13)C-doubly-labeled recombinant MUP-I were produced in a bacterial expression system and purified to homogeneity. Racemic 2-sec-butyl-4, 5-dihydrothiazole was produced synthetically. An equilibrium diffusion assay and NMR titration revealed that both enantiomers of the pheromone bind to the recombinant protein with a stoichiometry of 1 equiv of protein to 1 equiv of racemic pheromone. A micromolar dissociation constant and slow-exchange regime dissociation kinetics were determined for the pheromone-protein complex. (1)H, (15)N, and (13)C chemical shifts of MUP-I were assigned using triple resonance and (15)N-correlated 3D NMR experiments. Changes in protein (1)H(N) and (15)N(H) chemical shifts upon addition of pheromone were used to identify the ligand binding site. Several amide signals, corresponding to residues on one side of the binding site, were split into two peaks in the saturated protein-ligand complex. Similarly, two overlapping ligand spin systems were present in isotope-filtered NMR spectra of labeled protein bound to unlabeled pheromone. The two sets of peaks were attributed to the two possible chiralities of the pheromone. Intermolecular NOEs indicated that the orientation of the pheromone in the MUP-I binding cavity is opposite to that modeled in a previous X-ray structure.}
}

% 10404164 
@Article{pmid10404164,
   Author="Matsumura, I.  and Wallingford, J. B.  and Surana, N. K.  and Vize, P. D.  and Ellington, A. D. ",
   Title="{{D}irected evolution of the surface chemistry of the reporter enzyme beta-glucuronidase}",
   Journal="Nat. Biotechnol.",
   Year="1999",
   Volume="17",
   Number="7",
   Pages="696--701",
   Month="Jul",
   Abstract={The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited due to loss of activity during tissue fixation by glutaraldehyde or formaldehyde. We have directed the evolution of a GUS variant that is significantly more resistant to both glutaraldehyde and formaldehyde than the wild-type enzyme. A variant with eight amino acid changes was isolated after three rounds of mutation, DNA shuffling, and screening. Surprisingly, although glutaraldehyde is known to modify and cross-link free amines, only one lysine residue was mutated. Instead, amino acid changes generally occurred near conserved lysines, implying that the surface chemistry of the enzyme was selected to either accept or avoid glutaraldehyde modifications that would normally have inhibited function. We have shown that the GUS variant can be used to trace cell lineages in Xenopus embryos under standard fixation conditions, allowing double staining when used in conjunction with other reporters.}
}