Improved RNA polymerase ribozymes through functional selection

High-fidelity RNA polymerase ribozymes are imperative for the self-replication of functional RNAs, which is fundamental to RNA world hypothesis. Since the early 1990s, extensive efforts have being made by several research groups to evolve the class I polymerase ribozyme for improved sequence generality and tolerance of complex structures. Currently, the most proficient variant is the 24-3 polymerase (Horning & Joyce, PNAS, 2016), which can synthesize RNAs as complex as tRNA. This variant was obtained by in vitro evolution by selecting for its ability to synthesize functional RNA aptamers. The enzyme also has been shown to catalyze the exponential amplification of short RNA templates. However, the enzyme is prone to wobble misincorporation and is still unable to traverse complex sequence and structural motifs. Further evolution with increased selection pressure is expected to overcome these limitations. Because ribozymes are generally more sensitive to mutations, selection based on the synthesis of functional ribozymes rather than functional aptamers is expected to exert higher stringency. Efforts are ongoing in these directions. In this context, newly-described biorthogonal reactions also are being explored to manipulate the functional RNA molecules as part of the selection process.

David Horning, PhD – Evolving ribozymes to assemble aptamers

Katrina Tjhung – Evolving cross-chiral polymerase ribozymes