Mechanistic studies of cation catalysis in nonenzymatic RNA primer extension

Abstract

The origin of life is believed to have centered around RNA acting as both genetic information and catalyst. Several decades into research around the RNA world hypothesis, however, life has yet to be created synthetically. This is in part due to the difficulty of transitioning from the prebiotic synthesis of RNA monomers to RNA oligonucleotides, and the subsequent replication of RNA. Only once RNA can undergo replication nonenzymatically and react to the lengths of ribozymes can the full potential of this theory be evaluated. While much has been learned mechanistically about nonenzymatic RNA copying in the last several years, a particular gap in knowledge surrounds the role of metal ion catalysis in nonenzymatic RNA primer extension, due to the weak and transient nature of this catalytic interaction. This dissertation aims to characterize the nature of the interactions of cations in primer extension. In Chapter 1, the history of prebiotic chemistry is reviewed, as is what is known about RNA-cation interactions. In Chapter 2, I describe the metal ion, kinetics, and binding studies that support the model of 3′-OH nucleophilic activation from an inner-sphere contact with a catalytic cation. I additionally show the sequence, structural, and cation dependent effects of metal ion interactions in primer extension. I further support the previously published evidence shown indicating the 3′-OH is deprotonated before the rate-limiting step through kinetic isotope effects, and exclude the role of a metal-bound hydroxide species. Lastly, I measure the relative binding of metal ions as affected by the distance of the reacting bridged dinucleotide, suggesting an interaction with an oxygen of the bridged dinucleotide and the catalytic metal ion. In Chapter 3, I further validate this metal-bridged dinucleotide interaction as being relevant for primer extension through thiophosphoroimidazolide studies and metal rescue. We show that only one product is observed in the presence of Mg2+ in crystallo, regardless of starting material identity. Due to racemization of the starting material over the time scale of crystallography, this observation supports that only one diastereomer is reactive for primer extension, supporting a metal ion interaction with one specific prochiral oxygen on phosphorous, and an SN2-like mechanism for primer extension. Through this mechanistic understanding, the stereochemistry of the diastereomers can be assigned, and the coordination geometry of the catalytic metal ion is proposed, allowing chelation development to be performed intelligently. In Chapter 4, we address the possibility of Mn2+ as a prebiotically plausible catalyst in the RNA world, and find it improves ligation, primer extension rates (homo-polymeric and mixed template), and reaction yields, despite an increase in hydrolysis of reacting bridged dinucleotide. However, the increase in reactivity in the presence of Mn2+ results in a loss in fidelity at comparable concentrations of metal ion used for Mg2+, likely due to increased competition of activated monomers (which react with lower fidelity), supporting pre-existing hypotheses on fidelity for nonenzymatic primer extension. In sum, the results in this dissertation show the importance of metal ions in nonenzymatic primer extension, provide experimental results to support a model of cation catalysis, and provide a scaffold for designing co-catalysts to better enable metal ion-RNA interactions to improve nonenzymatic primer extension.