Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

Structure-Activity Relationships in Nonenzymatic Template-Directed RNA Synthesis.

The template-directed synthesis of RNA played an important role in the transition from prebiotic chemistry to the beginnings of RNA based life, but the mechanism of RNA copying chemistry is incompletely understood. We measured the kinetics of template copying with a set of primers with modified 3'-nucleotides and determined the crystal structures of these modified nucleotides in the context of a primer/template/substrate-analog complex. pH-rate profiles and solvent isotope effects show that deprotonation of the primer 3'-hydroxyl occurs prior to the rate limiting step, the attack of the alkoxide on the activated phosphate of the incoming nucleotide. The analogs with a 3 E ribose conformation show the fastest formation of 3'-5' phosphodiester bonds. Among those derivatives, the reaction rate is strongly correlated with the electronegativity of the 2'-substituent. We interpret our results in terms of differences in steric bulk and charge distribution in the ground vs. transition states.

Template-Directed Catalysis of a Multistep Reaction Pathway for Nonenzymatic RNA Primer Extension.

Before the advent of polymerase enzymes, the copying of genetic material during the origin of life may have involved the nonenzymatic polymerization of RNA monomers that are more reactive than the biological nucleoside triphosphates. Activated RNA monomers such as nucleotide 5'-phosphoro-2-aminoimidazolides spontaneously form an imidazolium-bridged dinucleotide intermediate that undergoes rapid nonenzymatic template-directed primer extension. However, it is unknown whether the intermediate can form on the template or only in solution and whether the intermediate is prone to hydrolysis when bound to the template or reacts preferentially with the primer. Here we show that an activated monomer can first bind the template and then form an imidazolium-bridged intermediate by reacting with a 2-aminoimidazole-activated downstream oligonucleotide. We have also characterized the partition of the template-bound intermediate between hydrolysis and primer extension. In the presence of the catalytic metal ion Mg2+, >90% of the template-bound intermediate reacts with the adjacent primer to generate the primer extension product while less than 10% reacts with competing water. Our results indicate that an RNA template can catalyze a multistep phosphodiester bond formation pathway while minimizing hydrolysis with a specificity reminiscent of an enzyme-catalyzed reaction.