Simulations predict preferred Mg²⁺ coordination in a nonenzymatic primer-extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg²⁺ are required for efficient primer extension, but the mechanism by which Mg²⁺ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg²⁺ in facilitating the deprotonation of the primer 3'-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg²⁺ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg²⁺ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg²⁺ ion with both O3' of the terminal primer nucleotide and the pro-S_p nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3' and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg²⁺ binding that is most relevant to catalysis of primer extension.