Significance: The replication of RNA without the aid of evolved enzymes may have enabled the inheritance of useful molecular functions during the origin of life. Template-directed RNA copying is a posited step in RNA replication. Key steps on the path to copying of RNA templates have been studied in isolation, including chemical nucleotide activation, generation of a key reactive intermediate, and template-directed polymerization. Here we report a prebiotically plausible scenario under which these reactions can happen together under mutually compatible conditions. Thus, this pathway could potentially have operated in nature without the complicating requirement for exchange of materials between distinct environments.

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.