Effects of Chemically Activated L-Nucleotides on Nonenzymatic RNA Copying

It has long been known that L-nucleotides inhibit the nonenzymatic copying of D-RNA templates, suggesting that nucleotide homochirality must have been attained before the emergence of RNA replication and the Origin of Life. Recent advances in nonenzymatic RNA copying, including the use of 2-aminoimidazole–activated monomers, and the elucidation of a reaction mechanism involving 2-aminoimidazolium-bridged dinucleotides, have inspired us to revisit the effects of L-nucleotides on the copying of D-RNA. We find that 2-AI-activated D- and L-nucleotides form heterochiral bridged dinucleotides at a slightly faster rate than homochiral bridged dinucleotides. 2-AI-bridged homochiral L/L dinucleotides bind D-RNA primer-template complexes with a higher Km than D/D bridged dinucleotides and lead to much slower D-RNA primer extension. Heterochiral bridged dinucleotides can bind surprisingly well to D-RNA primer-template complexes but react slowly, leading to competitive inhibition. X-ray crystallography reveals that L-nucleotides bind to the template by standard base pairing, but that the reactive phosphate is not in a productive orientation for phosphodiester bond formation. Even more strikingly, an L-nucleotide at the 3′ end of a D-RNA primer is a chain terminator. X-ray crystallography shows that the 3′-hydroxyl of the terminal L-nucleotide faces away from the phosphate of the incoming reactive substrate, explaining the failure of continued primer extension. We also show that L-nucleotides can be incorporated in a D-RNA stem-loop via loop-closing ligation, removing them from the replicating pool. Together, our results provide a kinetic and structural framework for understanding why a high level, if not complete, homochirality is required for nonenzymatic RNA replication.