Nonenzymatic RNA replication in a mixture of 'spent' nucleotides

Nonenzymatic template-directed replication would have been affected by co-solutes in a heterogeneous prebiotic soup due to lack of enzymatic machinery. Unlike in contemporary biology, these reactions use chemically activated nucleotides, which undergo rapid hydrolysis forming nucleoside monophosphates ('spent' monomers). These co-solutes cannot extend the primer but continue to base pair with the template, thereby interfering with replication. We, therefore, aimed to understand how a mixture of 'spent' ribonucleotides would affect nonenzymatic replication. We observed the inhibition of replication in the mixture, wherein the predominant contribution came from the cognate Watson-Crick monomer, showing potential sequence dependence. Our study highlights how nonenzymatic RNA replication would have been directly affected by co-solutes, with ramifications for the emergence of functional polymers in an RNA World.