eLife 7, e36422 (2018)

Nonenzymatic RNA polymerization can occur through template-directed primer extension, in which nucleotide-5′-phosphoro-2-aminoimidazolides (2-AIpN) are suitable reactive monomers, and an imidazolium-bridged dinucleotide forms as an intermediate. Zhang et al. have now developed an approach for time-resolved crystallography studies on this system. The RNA primer–template complex was crystallized with nonreactive dGMP bound to the template-strand overhang of the RNA duplex. The crystals were then soaked with reactive 2-AIpG, which diffused into the large solvent channels and replaced the dGMP within minutes. Molecular snapshots of primer extension were obtained by freezing the crystals at different time points. First, two 2-AIpG molecules base paired with the two-nucleotide template overhang of the RNA duplex and formed the dinucleotide intermediate. Next, a phosphodiester bond formed between the primer 3′-hydroxyl group and the 5′-phosphate of the nucleotide in the +1 position. The dinucleotide binds in a pre-organized manner with a shorter distance to the primer than the monomer, explaining the higher reactivity of the primer for the dinucleotide. This method may also be useful for studying the role of metal ions during primer extension.