Structure–Activity Relationships in Nonenzymatic Template‐Directed RNA Synthesis

The template‐directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3′‐nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate‐analog complex. pH‐rate profiles and solvent isotope effects show that deprotonation of the primer 3′‐hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3E ribose conformation show the fastest formation of 3′–5′ phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2′‐substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states. We combine X‐ray crystallography and kinetic measurements of modified nucleic acid constructs to determine the requirements for efficient template copying. The 3E conformation of the furanose rings is preferred in the formation of 3′–5′ phosphodiester bonds. Our findings may explain why RNA is a privileged substrate amongst other plausible candidates.