Mg²⁺ Catalyzes Nonenzymatic RNA Primer Extension through a Concerted Outer-Sphere Mechanism

The nonenzymatic replication of the primordial genetic material was an essential stage in the origin of life. One intensively studied model for this process is the template-directed extension of an RNA primer with imidazolium-bridged dinucleotide substrates. This reaction is catalyzed by divalent metal ions such as Mg²⁺, but the mechanism of catalysis remains poorly understood. We have utilized classical molecular dynamics together with hybrid quantum mechanics/molecular mechanics to investigate the catalytic role of Mg²⁺. We used these approaches to compute the free energy landscape along key reaction coordinates and to quantify the impact of Mg²⁺ coordination at each step of the primer extension reaction. The presence of Mg²⁺ in the reaction center significantly lowers the pK_a of the nucleophilic 3'-OH group and reduces the probability of O2' attack. Based on the energetics of potential reaction pathways, our results suggest a preferred mechanism in which Mg²⁺ becomes outer-sphere coordinated to the oxygen of the 3'-hydroxyl group of the extending primer followed by concerted proton transfer and nucleophilic attack on the phosphate of the incoming nucleotide. Our results demonstrate the dual structural and electronic roles of Mg²⁺ in catalysis and provide insights that may inform the search for metal ion chelators that further enhance nonenzymatic primer extension.