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 to different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion to both O3′ of the terminal primer nucleotide and the pro-Sp non- bridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to preorganize 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. Our simulations lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension. STATEMENT OF SIGNIFICANCE Prior to the evolution of complex enzymes, the replication of genetic material must have relied on nonenzymatic mechanisms. Nonenzymatic RNA template copying can be achieved through the extension of a primer by reaction with a 2-aminoimidazole (2AI) bridged dinucleotide in the presence of Mg2+. Despite progress in understanding the mechanism of this reaction, the catalytic role of Mg2+ remains poorly understood. Here, we present a series of molecular dynamics simulations of a model RNA primer-extension complex in different potential reactive conformations. We find that one configuration of both the 2AI moiety and coordination state of the Mg2+ promotes a geometry that is most favorable to reaction, suggesting a potential structural role for Mg2+ and providing insights to guide future experiments.