Trinucleotide building blocks enable exponential ribozyme-catalysed RNA replication and open-ended growth of diverse RNA sequence pools

Abstract

RNA replication is considered a crucial stage in the origins of life. However, both enzymatic and non-enzymatic RNA replication cycles are impeded by the "strand separation problem" (SSP), a form of product inhibition arising from the extraordinary stability of RNA duplexes and their rapid kinetics of reannealing. Here we show that RNA trinucleotide triphosphates (triplets) can overcome the SSP by binding to and kinetically trapping dissociated RNA strands in a single-stranded form, while simultaneously serving as substrates for RNA replication by a triplet polymerase ribozyme (TPR). This enabled exponential replication of both (+) and (-) strands of double-stranded RNAs by the TPR when driven by coupled pH and freeze-thaw cycles. We demonstrate replication of a fragment of the ribozyme itself, and open-ended amplification of random RNA sequence pools over >70 cycles, with emergence of partial, distributive TPR self-replication and triplet codon drift towards a primordial genetic code. One-sentence summaryRNA trinucleotide substrates together with simple physicochemical cycles enable RNA-catalysed replication of double-stranded RNA and partial, distributive self-replication of an RNA polymerase ribozyme.