By putting a large pool of random RNA sequences through a process of directed mutation, choosing and enriching those sequences that best perform some pre-defined function, researchers are now creating artificial ribozymes. They start with an initial pool of around 1015 different RNA fragments, each able to occupy hundreds of random positions on a strand. Then the researchers link these random-sequence pool molecules to a specific substrate and select those that convert the substrate to a desired product. The selected molecules are then amplified using protein replicases, and this selection-amplification procedure is repeated until sequences with the desired activity dominate the pool.
By using this method, researchers have created a whole host of new ribozymes that catalyse a variety of reactions, such as RNA ligation (joining RNA units together with a phosphodiester bond), RNA phosphorylation, RNA branch formation, and amide and peptide bond formation - all important biochemical reactions that are catalysed by proteins in today's cells. However, the development of this method has been a decidedly mixed blessing for the RNA world hypothesis. As Bartel and Unrau explain: 'Before the technology of in vitro selection existed, it was easy to proclaim boldly that RNA could catalyse the reactions required in the RNA world - no one expected experimental verification. However, now the onus is not merely to propose a key reaction of the RNA world, but also to propose an RNA molecule that can perform such a reaction'.
The kind of proof that the RNA world hypothesis really needs is the creation of an RNA molecule that can replicate either itself or another RNA molecule, the kind of self-replicating ribozyme that really could have performed the roles of both DNA and enzymes in early life. Unfortunately, this has not yet happened. Ribozymes have been created that perform different aspects of that function, but not one that performs all of them. 'Three key features of an RNA replicase currently reside in three different ribozymes and reactions', say Bartel and Unrau. 'One efficiently catalyses the proper chemistry, another uses nucleoside triphosphates [the individual units of RNA] in a templated fashion, and the third recognises an RNA duplex without regard for sequence. To prove the replicase assumption of the RNA world hypothesis, these features must be united into a single ribozyme'.
Bartel and Unrau managed to produce a ribozyme that goes some way towards this goal. In a paper in Nature in 1998 (395, 260), they reported that they had created an RNA molecule with the ability to catalyse the formation of a glycosidic bond joining a ribose sugar to a base (uracil) to make a nucleotide, the main building block of DNA and RNA. Nevertheless, the construction of a self-replicating RNA molecule, even if possible, is still a long way off.
More disconcerting still for proponents of the RNA world hypothesis is the fact that even if one of these is eventually created it will not necessarily mean that an RNA strand with that particular sequence performed the same function on the early Earth. Indeed, it wouldn't even prove the validity of the RNA world hypothesis. 'Even if ribozymes for all the essential activities of an RNA world were generated and assembled into RNA-based life, this would only show that the fundamental properties of RNA are compatible with the "RNA world" scenario', say Bartel and Unrau.
As well as being hard to prove, the RNA world hypothesis will also be hard to disprove. Only a minute fraction of RNA sequences can be sampled in an experiment, so just because a sequence that performs a certain catalytic function can't be found, doesn't mean that there isn't one available.
Other evidence can come from 'molecular fossils', putative remnants of the RNA world that are still active in modern-day cells. The seven ribozymes that have been discovered so far are examples of these 'fossils'; another has turned up from recent studies of the structure of the ribosome, the cellular component where proteins are manufactured. These studies have shown that a large proportion of the ribosome is constructed from RNA, meaning that the component's structure may have remained largely unchanged from the RNA world.
Even so, hard proof of the RNA world hypothesis is probably not going to be immediately forthcoming. What may turn out to be easier to prove or disprove are the underlying assumptions on which the hypothesis is based.
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