Enhanced Fidelity and Sequence Generality of an RNA Polymerase Ribozyme
TL;DR
By applying stringent selection pressure over the course of 33 successive rounds of evolution, both the fidelity and sequence generality of the polymerase were greatly improved and the improved polymerase is able to synthesize a broad range of RNA products, including those that require copying templates that are highly structured or contain sequences that previously were difficult to copy.
Abstract
Directed evolution has been used to develop RNA enzymes (ribozymes) that catalyze the RNA-templated polymerization of RNA. The more advanced polymerases can synthesize long RNA products, but have shortcomings with regard to copying accuracy and template sequence generality, which are impediments to using the polymerase to replicate and evolve other functional RNAs. By applying stringent selection pressure over the course of 33 successive rounds of evolution, both the fidelity and sequence generality of the polymerase were greatly improved. When synthesizing large functional RNAs such as the class I ligase ribozyme, the fidelity improved from 86% to 94% per nucleotide, due to both reduced incorporation of mismatched nucleotides and reduced propensity to extend partial-length products that have mismatched termini. The improved polymerase is also able to synthesize a broad range of RNA products, including those that require copying templates that are highly structured or contain sequences that previously were difficult to copy.