High-Fidelity Templated Ligation of RNA via 2′,3′-cyclic Phosphate

Abstract

The templated ligation of oligonucleotides offers a mode of replication in an RNA world. The 2′,3′-cyclic phosphate (>P) is a prebiotically available activation group for RNA and the product of backbone hydrolysis. Using gel electrophoresis and liquid chromatography, we found that the templated ligation of RNA with >P activation proceeds in alkaline (pH 9-11) low-salt aqueous solutions with 1 mM MgCl2 in temperatures ranging from 20 to 25 °C within a few days. Under the optimum conditions of pH 10 and 5 °C, the ligation yielded 40% after 7 days. No additional catalysts were required. In contrast to previous reports, we found an equimolar mixture of 2′-5′ and 3′-5′ linked oligomers in the used conditions. We probed the nucleotide specificity at the ligation site and found that one mutation reduced the ligation yield by 82-92%. We extrapolated these results to a per-nucleotide replication fidelity of 95-98% when ligating 4- to 6-mers. With splinted oligomers, five ligations created a 96 mer strand, demonstrating a possible assembly pathway for long ribozymes. With the low salt requirements, strand separation will be compatible with the ligation conditions using non-equilibrium settings. The findings suggest that templated ligation mediated by 2′,3′-cyclic phosphate in alkaline conditions offer a slow, but precise replication and elongation reaction for RNA on early Earth.