On ribozymes

Hooray, it’s the end of the term. And what better way to relax than to jet off to a conference filled with physicists and historians and philosophers of science. What? Isn’t that what you’d do? Well, being here at the Seven Pines Symposium on the Origins of Life has nonetheless been both relaxing and intellectually stimulating. And it’s been a chance for me to think a bit about ribozymes, which I haven’t done in awhile. For those of you who may have read my previous entry “On Technological Surprise,” ribozymes are of course RNA enzymes.

A case can be made that ribozymes were our first catalysts. Looking forward from origins, it has been hypothesized that the earliest living system was likely a replicating nucleic acid. Looking backwards from modern life, the molecular fossils that live in our cells, such as cofactors and the ribosome, are made of nucleotides or RNA. Therefore, it is reasonable to conclude that there was once a living system (not “life,” see “On Origins”) whose metabolism, including its enzymes, was made largely of RNA. This is the so-called ribo-organism of the so-called RNA world. This organism committed suicide by inventing translation, but that’s another story.

While Crick and Orgel inferred the existence of the RNA world at least as early as 1968, it was indeed Tom Cech’s and Sydney Altman’s technologically surprising discovery of catalytic RNA that gave form to the fantasy. Since the identification of natural ribozymes, there have been multiple attempts to create artificial ribozymes by directed evolution. Many of these attempts have been surprisingly successful, and have yielded RNA molecules with catalytic properties that include the ability to make and break carbon bonds, carry out redox reactions, and of course rearrange bonds to phosphate like nobody’s business.

One of the earliest and best selections of a ribozyme de novo was carried out by David Bartel. Back when he was a lanky graduate student as opposed to a suave member of the Whitehead, David was the only one bold enough to select for ligase activity from a completely random sequence pool. His technical excellence resulted in multiple, different catalysts, one of which has proven to be particularly interesting, the Bartel Class I ligase. This enduring beast is especially interesting in that it is one of the few examples of a scientific miracle. The relative information content of this very fast ligase can be determined, and it turns out that its representation in its original random sequence pool should have been on the order of once in every 10^19 sequences. The problem is that the pool itself was on the order of only about 10^15 sequences. Therefore, either David Bartel was the luckiest graduate student in the world (a distinct possibility), or else … the ribozyme was but one of many other, similarly complex ligases. Should the tape of life (or, well, the tape of graduate student servitude) ever be run again, a different but equally complex ribozyme would emerge from the pool. And this is what you should tell Creationists who insist that histones couldn’t have evolved by accident because they are so highly conserved. Selection, whether natural or artificial, yields up functions, not particular sequences.

The Bartel ligase has since been on many wonderful adventures, ending up for awhile with Gerry Joyce and his group in an experiment that proved that ligase function could be selected continuously. If you squint, and substitute “sugar and minerals” with “nucleotides and enzymes,” the ribozyme ‘grows’ and evolves in the presence of its foodstuffs the same way a bacteria would grow and evolve in a flask.

The Bartel ligase was such a fertile and versatile system that for awhile there were attempts to make it act like a polymerase, not just ligating a single oligonucleotide substrate to itself, but rather serially ligating multiple nucleotides (stop me before I ligate again!). If only it could be made to catalyze a sufficient number of such additions, it could … replicate itself! This would be the primordial Xeroxase, the enzyme that one could point to and proudly say, “That’s life!” (even if there is no such thing). Alas, try as they might, researchers could not make the Bartel ligase catalyze more than a few turns of a helix, reproducing only a shadow of itself.

Until recently. Until now. When Holliger and his co-workers have come along and done the remarkable (Science, 332:209): in a directed evolution experiment that was such a technical tour-de-force that it rivals the selection of the original ribozyme itself, these researchers made the ribozyme replicate … 95 nucleotides. Of a very special template. Still! Almost there! Actually, roughly halfway, based on the length of the parental ribozyme. Given this breakthrough, ribozyme-catalyzed replication (the Xeroxase) may almost be at hand (go Jared!).

Here’s another sort of funny thing about the Bartel ligase, something that is seldom remarked upon: it doesn’t like to be mutated. More properly, it seems to inhabit a very narrow peak on a sequence landscape, and many mutations can knock it from its perch. So, over various selections by various folks, it has only grudgingly changed, and even Wochner et al. (2011) got it to budge by only a few (4) mutations.

This is decidedly not like the sequence landscapes that one observes in natural selection, where biopolymers often move about on large, neutral networks, happily collecting neutral mutations. This difference may be due to the difference in origins: the Bartel ligase emerged from a completely randomized population; natural biopolymers typically evolve one mutation at a time. It’s sort of the difference between betting a gut shot straight in poker, versus having many outs. Most of the time, in trying to hit the straight you go bust, while you may make a lesser hand more easily.

And here’s the payoff, so to speak: because this ribozyme was so very finicky, I wasn’t sure it would ever go anywhere. I’ve often thought it was a beautiful evolutionary dead end. But Holliger and his crew breathed life into it, and revived all of our dreams of making a Xeroxase.

This difference between perception and reality illustrates several important things: (1) I frequently have terrible instincts. We knew that already. (2) Selection can yield amazing products … but only if done with technical excellence. (3) There may be exquisitely narrow fitness peaks with extraordinary properties. It’s this latter that has some biodefense relevance. The role of directed evolution in the production of biosensors and remediation catalysts and whatnot comes and goes, as do most fads. But there may not be natural catalysts that can robustly perform in military applications, and while we continue to wait for the dominance of computational design, directed evolution remains one of the few ways to plumb the nooks and crannies of the world outside biology.


- originally posted on Sunday, May 22nd, 2011.