Enhanced Nonenzymatic RNA Copying and Replication

Abstract

The nonenzymatic RNA copying and replication are believed to have played a crucial role in the transition from prebiotic chemistry to the emergence of ribozyme-catalyzed RNA replication to construct protocells. Most of the earlier research on this subject did not fully consider how nonenzymatic RNA copying and replication could benefit from the complexity of materials available on prebiotic Earth, which likely contained a diverse pool of nucleotides and oligonucleotides. My dissertation seeks to address this gap by investigating the limitations of current model systems and proposing new strategies for nonenzymatic copying and replication. I start by providing kinetic explanations for the sequence biases and low yields in mixed RNA template copying. Based on these kinetic observations, I propose two strategies to enhance unbiased nonenzymatic copying by utilizing thiolated nucleotides and monomer-bridged-oligonucleotides. Following these proposals, I demonstrate how 2-thiouridine (s2U) substitutions can lead to fast nonenzymatic copying with good fidelity, even with the recent discovery of an unusual s2U:s2U base pair that stabilizes RNA duplexes. As an alternative pathway, I next present how prebiotically plausible methylisocyanide- mediated chemistry can be used to activate short oligonucleotides and catalyze fast nonenzymatic RNA copying. Finally, I introduce a virtual circular genome (VCG) model as a means of going beyond template copying to achieve indefinite cycles of nonenzymatic RNA replication. In this model, the genome is represented by a collection of short oligonucleotides that can map onto two complementary virtual circular sequences. Replication is driven by nonenzymatic templated extension of kinetically trapped partially base-paired strands, followed by rearrangement of these configurations to enable continuous oligonucleotide elongation. I show that the extension in this model is greatly enhanced when all the VCG oligonucleotides are activated. Furthermore, I demonstrate the nonenzymatic extension in the VCG system when encapsulated inside a model protocell. The new observations and models presented in this dissertation for both nonenzymatic RNA copying and replication lay the foundation for further research on the assembly of a self-replicating protocell.