Ribozyme-catalysed RNA synthesis using triplet building blocks.

James Attwater,Edoardo Gianni,Alexey S Morgunov,Aditya Raguram,Philipp Holliger

doi:10.7554/elife.35255

Abstract

RNA-catalyzed RNA replication is widely believed to have supported a primordial biology. However, RNA catalysis is dependent upon RNA folding, and this yields structures that can block replication of such RNAs. To address this apparent paradox, we have re-examined the building blocks used for RNA replication. We report RNA-catalysed RNA synthesis on structured templates when using trinucleotide triphosphates (triplets) as substrates, catalysed by a general and accurate triplet polymerase ribozyme that emerged from in vitro evolution as a mutualistic RNA heterodimer. The triplets cooperatively invaded and unraveled even highly stable RNA secondary structures, and support non-canonical primer-free and bidirectional modes of RNA synthesis and replication. Triplet substrates thus resolve a central incongruity of RNA replication, and here allow the ribozyme to synthesise its own catalytic subunit '+' and '-' strands in segments and assemble them into a new active ribozyme.

Highlights

The premise that some RNA sequences can catalyse and template their own replication reciprocally synthesizing their own ‘+’ and ‘–’ strands - underpins current thinking about early genetic systems (Crick, 1968; Orgel, 1968; Szostak et al, 2001)
We set out to explore the potential of short RNA oligonucleotides as substrates for RNA-catalyzed RNA replication
In order to assess the fidelity of triplet polymerase ribozymes of widely differing activity, we identified the triplets incorporated from random pppNNN triplet pools using 12 different compositionally representative N0N0N0 triplet sequences as templates

Summary

Introduction

The premise that some RNA sequences can catalyse and template their own replication reciprocally synthesizing their own ‘+’ and ‘–’ strands - underpins current thinking about early genetic systems (Crick, 1968; Orgel, 1968; Szostak et al, 2001). Even the most highly-evolved RPRs (Horning and Joyce, 2016) are substantially impeded by template secondary structures. The strong inhibitory role of this central feature of RNA leads to an antagonism between the degree to which an RNA sequence is able to fold into a defined three-dimensional structure to encode function (such as catalysis) and the ease with which it can be replicated (Boza et al, 2014).

Methods

Results

Conclusion