Nonenzymatic RNA copying with a potentially primordial genetic alphabet

Abstract Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U, because of the stronger G:C vs. A:U base pair, and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s 2 U) and inosine (I) form by deamination of 2-thiocytidine (s 2 C) and A respectively. We used thermodynamic and crystallographic analyses to compare the I:s 2 C and A:s 2 U base pairs. We find that the I:s 2 C base pair is isomorphic and isoenergetic with the A:s 2 U base pair. The I:s 2 C base pair is weaker than a canonical G:C base pair, while the A:s 2 U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s 2 U, s 2 C, I and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s 2 C and s 2 U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s 2 U, s 2 C, I and A, and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. Significance Statement A long-standing challenge in primordial nonenzymatic RNA copying chemistry is the biased incorporation of C and G over A and U due to differences in base pair strength. We hypothesized that 2-thiopyrimidine substitution could help overcome this bias since A:s 2 U is a stronger version of the A:U base pair, and I:s 2 C is a weaker version of the G:C base pair. This study explores the efficacy of a potentially primordial genetic alphabet consisting of s 2 U, s 2 C, A and I. Our results show that A:s 2 U and I:s 2 C pairs are isoenergetic and isomorphic. Our findings highlight the potential of this alternative genetic alphabet to yield a more balanced incorporation of all nucleotides, facilitating information propagation by nonenzymatic RNA copying during the origin of life.