The abiotic synthesis of ribonucleotides is thought to have been an essential step toward the emergence of the RNA world. However, it is likely that the prebiotic synthesis of ribonucleotides was accompanied by the simultaneous synthesis of arabinonucleotides, 2′-deoxyribonucleotides, and other variations on the canonical nucleotides. In order to understand how relatively homogeneous RNA could have emerged from such complex mixtures, we have examined the properties of arabinonucleotides and 2′-deoxyribonucleotides in nonenzymatic template-directed primer extension reactions. We show that nonenzymatic primer extension with activated arabinonucleotides is much less efficient than with activated ribonucleotides, and furthermore that once an arabinonucleotide is incorporated, continued primer extension is strongly inhibited. As previously shown, 2′-deoxyribonucleotides are also less efficiently incorporated in primer extension reactions, but the difference is more modest. Experiments with mixtures of nucleotides suggest that the coexistence of riboand arabinonucleotides does not impede the copying of RNA templates. Moreover, chimeric oligoribonucleotides containing 2′deoxy-or arabinonucleotides are effective templates for RNA synthesis. We propose that the initial genetic polymers were random sequence chimeric oligonucleotides formed by untemplated polymerization, but that template copying chemistry favored RNA synthesis; multiple rounds of replication may have led to pools of oligomers composed mainly of RNA.

SignificanceThe RNA world hypothesis assumes the abiotic synthesis of nucleotides, as well as their participation in nonenzymatic RNA replication. Whereas prebiotic syntheses of the canonical purine nucleotides remain inefficient, a prebiotically plausible route to the 8-oxo-purines has been reported. Although these noncanonical purine nucleotides are known to engage in non-Watson–Crick pairing with their canonical purine counterparts, their behavior in nonenzymatic RNA copying has not been evaluated. Our study indicates that none of the 8-oxo-purines behaves as a suitable substrate for nonenzymatic RNA copying. However, inosine turns out to exhibit reasonable rates and fidelities in RNA copying reactions. We propose that inosine could have served as a surrogate for guanosine in the early emergence of life.

Non-enzymatic RNA self-replication is integral to the emergence of the ‘RNA World’. Despite considerable progress in non-enzymatic template copying, demonstrating a full replication cycle remains challenging due to the difficulty of separating the strands of the product duplex. Here, we report a prebiotically plausible approach to strand displacement synthesis in which short ‘invader’ oligonucleotides unwind an RNA duplex through a toehold/branch migration mechanism, allowing non-enzymatic primer extension on a template that was previously occupied by its complementary strand. Kinetic studies of single-step reactions suggest that following invader binding, branch migration results in a 2:3 partition of the template between open and closed states. Finally, we demonstrate continued primer extension with strand displacement by employing activated 3′-aminonucleotides, a more reactive proxy for ribonucleotides. Our study suggests that complete cycles of non-enzymatic replication of the primordial genetic material may have been facilitated by short RNA oligonucleotides.