Enhanced Fidelity and Sequence Generality of an RNA Polymerase Ribozyme

Long before complex life with DNA and proteins existed, scientists believe the first living systems were built entirely from a single molecule called RNA. In this ancient era, often called the "RNA world," RNA had to do double duty: it carried genetic instructions like a blueprint, and it acted as a machine to copy itself, like a factory worker. For life to grow and evolve, this RNA machine needed to be incredibly precise. If it made too many mistakes while copying its own instructions, the information would become garbled and useless, causing the system to collapse. However, creating an RNA molecule that can copy itself with high accuracy is a monumental challenge. While scientists have previously engineered RNA enzymes that can build other RNA strands, these early versions were clumsy. They could only copy simple, unstructured sequences and often made too many errors to sustain a living system. They were like a photocopier that could only handle blank paper or single lines of text, failing whenever the document became complex or folded into a shape.

A team of researchers at The Salk Institute has now engineered a new version of this RNA machine that overcomes these limitations. By subjecting the enzyme to a rigorous process of trial and error, they created a ribozyme, or RNA enzyme, that can copy complex, folded RNA strands with remarkable accuracy. This new machine, named 85h34, represents a significant leap forward. It can read difficult templates that were previously impossible to copy, and it makes far fewer mistakes than its predecessors. Most importantly, it can do this even when the chemical environment is made less favorable, a condition that forces the machine to be more careful. This achievement brings scientists one step closer to creating a self-replicating system that could evolve on its own, offering a glimpse into how life might have started from simple chemistry.

The journey to create this improved machine began with a previous version of the RNA polymerase, known as 52-2. While this earlier model could copy RNA, it struggled with accuracy, especially when the template was long or had a complex structure. When it tried to copy a specific RNA molecule called the class I ligase, which is 97 units long, it made so many errors that the resulting copies were almost entirely useless. To fix this, the researchers decided to put the enzyme under extreme pressure. They designed an experiment where the only way for the enzyme to survive and reproduce was if it successfully built a working copy of the class I ligase. If the enzyme made a mistake, the copy would not function, and that version of the enzyme would be discarded.

The researchers ran this selection process for 33 additional rounds of evolution. With each round, they made the task harder. They reduced the time the enzyme had to finish the job and, crucially, they lowered the concentration of magnesium ions in the solution. Magnesium acts as a helper for the chemical reaction, but by reducing its amount, the researchers forced the enzyme to work more efficiently and carefully. A lower concentration of magnesium also makes it harder for the enzyme to ignore mistakes, effectively punishing it for copying errors. Over these 33 rounds, the population of RNA enzymes changed dramatically. The researchers tracked these changes by sequencing the genetic code of the enzymes at every step, watching how different groups, or clades, of mutations rose and fell in popularity.

The data revealed a complex history of competition. Early in the process, one group of enzymes became dominant because it was very accurate, but it was too slow to finish the job in the shortened time limits. As the pressure increased, this group died out. Two other groups, which had been present in small numbers, began to thrive. One group improved its ability to handle the difficult, folded parts of the template, while the other became better at maintaining speed and accuracy under low magnesium conditions. These two groups carried different sets of beneficial mutations that were separated by large distances in the RNA sequence, making it nearly impossible for them to naturally combine their best traits through random recombination. To solve this, the researchers manually combined the best features from both groups into a single, hybrid enzyme.

This new hybrid, named 85h34, proved to be a powerhouse. It could synthesize the complex class I ligase with high yield, even in conditions where the older versions failed completely. When tested on a variety of RNA templates, including those with difficult sequences and folded structures, 85h34 consistently outperformed its predecessors. It could copy templates that contained long stretches of difficult-to-read sequences and could even unwind and copy through hairpin loops that caused the older enzymes to stall. Perhaps most impressively, 85h34 retained its ability to work when the magnesium concentration was reduced to just 10 millimolar, a level so low that no other RNA polymerase ribozyme had ever been able to function, provided that a molecular crowding agent (8% PEG8000) was added to the solution.

The accuracy of this new machine was measured by counting the errors it made during the copying process. The older 52-2 enzyme made mistakes about 14 times out of every 100 nucleotides it copied when working on the class I ligase. In contrast, the new 85h34 enzyme made errors only about 6 times out of every 100 nucleotides under standard conditions, and this number dropped to just 5 errors per 100 when working in the low magnesium environment. This improvement came from two sources: the new enzyme was better at picking the correct building blocks to start with, and it was much better at stopping itself from continuing if it had already made a mistake. By refusing to extend a flawed copy, it prevented the accumulation of errors in the final product.

The significance of this work lies in what it enables. For life to evolve, genetic information must be passed down with enough fidelity to preserve useful traits, but with enough variation to allow for innovation. The 85h34 polymerase has crossed a critical threshold where it can reliably copy complex functional RNAs without the information degrading into nonsense. This means that scientists can now use this enzyme to evolve other RNA molecules, creating a system where RNA can not only copy itself but also improve itself over time. The researchers suggest that this bootstrapping process, where a better copy machine creates better copies of itself, could eventually lead to a fully self-evolving RNA system. While the paper does not claim to have created life, it has constructed the most capable RNA copying machine to date, providing a robust tool to explore the chemical origins of life and the fundamental principles of evolution.