Joyce, G. F. | Search bioRxiv

bioRxiv2026

Enhanced Fidelity and Sequence Generality of an RNA Polymerase Ribozyme

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.

biochemistry↗

bioRxiv2021

Witnessing the Structural Evolution of an RNA Enzyme

An RNA polymerase ribozyme that has been the subject of extensive directed evolution efforts has attained the ability to synthesize complex functional RNAs, including a full-length copy of its own evolutionary ancestor. During the course of evolution, the catalytic core of the ribozyme has undergone a major structural rearrangement, resulting in a novel tertiary structural element that lies in close proximity to the active site. Through a combination of site-directed mutagenesis, structural probing, and deep sequencing analysis, the trajectory of evolution was seen to involve the progressive stabilization of the new structure, which provides the basis for improved catalytic activity of the ribozyme. Multiple paths to the new structure were explored by the evolving population, converging upon a common solution. Tertiary structural remodeling of RNA is known to occur in nature, as evidenced by the phylogenetic analysis of extant organisms, but this type of structural innovation had not previously been observed in an experimental setting. Despite prior speculation that the catalytic core of the ribozyme had become trapped in a narrow local fitness optimum, the evolving population has broken through to a new fitness locale, suggesting that further improvement of polymerase activity may be achievable.

biochemistry↗