There are plenty of unanswered questions about the origin of life on Earth. But the research community has largely reached consensus that one of the key steps was the emergence of an RNA molecule that could replicate itself. RNA, like its more famous relative DNA, can carry genetic information. But it can also fold up into three-dimensional structures that act as catalysts. These two features have led to the suggestion that early life was protein-free, with RNA handling both heredity and catalyzing a simple metabolism.

For this to work, one of the reactions that the early RNAs would need to catalyze is the copying of RNA molecules, without which any sort of heritability would be impossible. While we’ve found a number of catalytic RNAs that can copy other molecules, none have been able to perform a key reaction: making a copy of themselves. Now, however, a team has found an incredibly short piece of RNA—just 45 bases long—that can make a copy of itself.

Finding an RNA polymerase

We have identified a large number of catalytic RNAs (generically called ribozymes, for RNA-based enzymes), and some of them can catalyze reactions involving other RNAs. A handful of these are ligases, which link together two RNA molecules. In some cases, they need these molecules to be held together by a third RNA molecule that base pairs with both of them. We’ve only identified a few that can act as polymerases, which add RNA bases to a growing molecule, one at a time, with each new addition base pairing with a template molecule.

Obviously, there is some functional overlap between them, as you can think of a polymerase as ligating on one base at a time. And in fact, at the ribozyme level, there’s some real-world overlap, as some ribozymes that were first identified as ligases were converted into polymerases by selecting for this new function.

While this is fascinating, there are a few problems with these known examples of polymerase ribozymes. One is that they’re long. So long, in fact, that they’re beyond the length of the sort of molecules that we’ve observed forming spontaneously from a mix of individual RNA bases. This length also means they’re largely incapable of making copies of themselves—the reactions are slow and inefficient enough that they simply stop before copying the entire molecule.

Another factor related to their length is that they tend to form very complex structures, with many different areas of the molecule base-paired to one another. That leaves very little of the molecule in a single-stranded form, which is needed to make a copy.

Based on past successes, a French-UK team decided to start a search for a polymerase by looking for a ligase. And they limited that search in an important way: They only tested short molecules. They started with pools of RNA molecules, each with a different random sequence, ranging from 40 to 80 bases. Overall, they estimated that they made a population of 10¹³ molecules out of the total possible population of 10²⁴ sequences of this type.

These random molecules were fed a collection of three-base-long RNAs, each linked to a chemical tag. The idea was that if a molecule is capable of ligating one of these short RNA fragments to itself, it could be pulled out using the tag. The mixtures were then placed in a salty mixture of water and ice, as this can promote reactions involving RNAs.

After 11 rounds of reactions and tag-based purification, the researchers ended up with three different RNA molecules that could each ligate three-base-long RNAs to existing molecules. Each of these molecules was subjected to mutagenesis and further rounds of selection. This ultimately left the researchers with a single, 51-base-long molecule that could add clusters of three bases to a growing RNA strand, depending on their ability to base-pair with an RNA template. They called this “polymerase QT-51,” with QT standing for “quite tiny.” They later found that they could shorten this to QT-45 without losing significant enzyme activity.

Checking its function

The basic characterization of QT-45 showed that it has some very impressive properties for a molecule that, by nucleic acid standards, is indeed quite tiny. While it was selected for linking collections of molecules that were three bases long, it could also link longer RNAs, work on shorter two-base molecules, or even add a single base at a time, though this was less efficient. While it worked slowly, the molecule’s active half-life was well over 100 days, so it had plenty of time to get things done before it degraded.

It also didn’t need to interact with any specific RNA sequences to work, suggesting it had a general affinity for RNA molecules. As a result, it wasn’t especially picky about the sequences it could copy.

As you might expect from such a small molecule, QT-45 didn’t tolerate changes to its own sequence very well—nearly the entire molecule was important in one way or another. Tests that involved changing every single individual base one at a time showed that almost all the changes reduced the ribozyme’s activity. There were, however, a handful of changes that improved things, suggesting that further selection could potentially yield additional improvements. And the impact of mutations near the center of the sequence was far more severe, suggesting that region is critical for QT-45’s enzymatic activity.

The team then started testing its ability to synthesize copies of other RNA molecules when given a mixture of all possible three-base sequences. One of the tests included a large stretch in which one end of the sequence base-paired with the other. To copy that, those base pairs need to somehow be pried apart. But QT-45 was able to make a copy, meaning it synthesized a strand that was able to base pair with the original.

It was also able to make a copy of a template strand that would base pair with a small ribozyme. That copying produced an active ribozyme.

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