Investigating the Mechanism of Non-Enzymatic RNA Replication

Abstract

Before life became confoundingly complex it must have been simpler. In modern biology, polymerase enzymes leverage the molecular recognition of nucleic acids through Watson-Crick base pairs to efficiently synthesize genomic material. Templated RNA synthesis could have supported early life by enabling genomic replication in the absence of enzymatic catalysts. Despite encouraging early results, the model still has severe shortcomings: only some sequences are copied efficiently, multiple regioisomers are formed and RNA degradation is as fast as its synthesis. In my thesis work I show that 2-aminoimidazole, the leaving group used in non-enzymatic RNA copying, can be obtained prebiotically in similar conditions to those in which RNA is synthesized, suggesting a common origin for RNA synthesis and activation. 2-Aminoimidazole activated ribonucleotides enable faster RNA synthesis through the formation of an imidazolium bridged intermediate. I then show that the poor regioselectivity of RNA synthesis is overstated: fast reactions, in which the reactive intermediate can bind through Watson-Crick base-pairs to the template produce only the correct regioisomer. To further optimize the reaction, I set out to determine its mechanism. To this end, I developed a high-throughput assay for templated RNA synthesis and investigated the role of nucleic acid structure and of catalytic metal ions. I found that RNA is particularly adept at templated synthesis because of its preference for a specific conformation and that Mg ions catalyze RNA synthesis by forming a reactive anion. Lastly, RNA degradation is mitigated by templated synthesis, giving rise to a novel biopolymer: pyrophosphate linked RNA. My work adds additional support to the RNA world hypothesis while also revealing fundamental aspects of nucleic acid chemistry and developing the synthesis of alternative biopolymers. These findings point towards a mechanism of chemical selection that explains why RNA, and not its analogs, is present four billion years later.