According to the “RNA world” model of life's origin, RNA performed all of the operations that are essential to life. RNA alone passed on genetic information and catalyzed the reactions of basic metabolism; DNA and proteins were not in the picture. The RNA world hypothesis is an appealingly simple model for simple early life forms, as it allows the complex array of biochemical interactions among proteins, DNA, and RNA to evolve gradually.

Our current natural world no longer uses RNA enzymes that act on their own to perform most biological functions. To better understand ancient RNA enzymes, modern scientists have to rely on proxies, like engineered RNA "ribozymes" that have catalytic functions without the need for proteins. However, scientists have had trouble creating a proxy for the first self-replicating molecule, or even an RNA ribozyme that can copy an RNA that's long enough to have further biological functions. Aniela Wochner and her coauthors have overcome that difficulty. In a recent issue of Science, they report the creation of an RNA ribozyme that synthesizes complex RNAs, including RNAs that act as ribozymes and perform a biological function.

Previously, the leading RNA polymerase ribozyme, called R18, could only transcribe RNAs up to 14 bases long (as a frame of reference, R18 itself is about 196 bases long). It was also highly template-dependent, meaning it could only copy certain sequences of RNA. To establish early life on Earth, a ribozyme would need to be able to make a variety of RNA sequences of adequate length, including something long enough to synthesize itself. Wochner and her colleagues sought to engineer a superior RNA ribozyme by modifying R18.

First, they wanted to improve the interactions among the template RNA, the ribozyme, and the primer sequence that starts the copying. To make RNA, the ribozyme has to recognize the primer and the template, which base-pair with one another. Then, the ribozyme catalyzes the addition of new bases onto the primer, making an RNA sequence that is complementary to the template.

Scientists have proposed that the one end of the R18 ribozyme interacts with the primer and template. So, Wochner and her colleagues appended a random sequence into the 5’-end of R18 and selected for improved RNA polymerase activity.

They found one ribozyme (named C19) that did better than R18 on a specific, short template, but it didn't work well on longer templates. They further modified C19 by making truncated variants of its sequence and screened for improved activity on longer templates. They found one variant (the ribozyme tC19) that can extend primers by up to 95 bases with favorable templates.

The final obstacle was the fact that it only worked well on favorable template sequences—the researchers wanted a ribozyme that will be able to copy a diverse range of RNA templates, not just a few favorable ones. To find one, they made 50 million randomly mutated R18 sequences, did numerous rounds of selections, and found a combination of mutations that improved the recognition of diverse templates. They applied those mutations to the tC19 ribozyme, creating the RNA polymerase ribozyme tC19Z (198-bases long).

Ribozyme tC19Z synthesizes longer RNA sequences and can work with a greater range of primer-template combinations than any of the previous ribozymes. Wochner and her colleagues were able to use tC19Z to synthesize a minimal version of the hammerhead ribozyme (an RNA that binds to and cleaves an RNA substrate). The synthesized hammerhead ribozyme had catalytic activity, as it was able to cleave an RNA substrate at the expected location in the sequence.

Wochner and her coauthors have significantly expanded our abilities to engineer RNA polymerase ribozymes; however, further improvements are still necessary. For example, tC19Z probably cannot synthesize something of its own size in a reasonable amount of time. Nevertheless, it's impressive that the researchers were able to select for such drastic improvements on R18, as the sequence hasn't seen a significant upgrade since its creation almost a decade ago. Their work lead us closer to understanding ribozymes that could have existed in early Earth.

Science, 2011. DOI: 10.1126/science.1200752 (About DOIs)

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