Towards Constructing Informational Replicating Systems in Nonenzymatic Environments
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CitationZhang, Weicheng. 2018. Towards Constructing Informational Replicating Systems in Nonenzymatic Environments. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractTremendous efforts have been made in building an artificial protocell that could undergo self-replication and Darwinian evolution. Self-replicating genetic polymers, which serve as major components of protocells, have been a major focus of the origins of life research for many decades. Numerous genetic polymers, especially RNA, have been studied in search of a model that allows effective self-replication in nonenzymatic environments. In this thesis, we aim to discover new alternative genetic polymers with enhanced copying chemistry and explore optimal reaction conditions for efficient copying of RNA.
Chapter 1 summarizes the key findings of the behavior of different types of nucleic acids in nonenzymatic, template-directed polymerization or replication.
Chapter 2 describes the development of a new model system using morpholino nucleic acid (MoNA) to enhance the reaction speed of the nonenzymatic replication of oligonucleotides. The template-directed polymerization of activated ribonucleotide monomers is generally slow because the 3′ primer hydroxyl group has relatively weak nucleophilicity. Previous studies have shown that replacing the hydroxyl group with more nucleophilic amine group generally results in faster primer extension. Herein, we have chosen to study MoNA as it contains a cyclic secondary amine which is expected to be highly nucleophilic. In this study, we describe the synthesis of 2-methylimidazole activated MoNA monomers from their corresponding ribonucleotides 5′-monophosphates, as well as the synthesis of an RNA primer with a terminal MoNA nucleotide. We discover that activated G and C MoNA monomers enable more rapid and efficient extension of the morpholino-terminated primer on homopolymeric and mixed-sequenced RNA templates.
Chapter 3 describes the importance of the precise position of the hydroxyl at the 3′-end of the primer for the nonenzymatic template-directed primer extension. In this study, we aim to examine the effect of spatial displacement of the hydroxyl nucleophile. RNA primers terminating in either 2′-deoxy-guanosine or 3′-hydroxymethyl-2′,3′-dideoxyguanosine residues were synthesized and their rates in the primer extension reactions were compared. The decreased activity of the hydroxymethyl terminated primer suggests that the spatial preorganization of the deoxyribose and ribose sugars contributes significantly to the efficiency of nonenzymatic primer extension.
Chapter 4 presents a comprehensive analysis of the kinetic profile of nonenzymatic RNA primer extension, the thermodynamic favorability of duplex formation, and the rate of monomer hydrolysis in response to the presence of Mg2+, Mn2+, and (Co(NH3)63+. Multivalent metal ions are essential catalyst in the nonenzymatic replication of RNA. Metal ions play multiple key roles in nonenzymatic primer extension, including stabilizing the RNA duplex and activating the nucleophile and/or the electrophile at the reaction site. In this chapter, we demonstrate that the application of cooperative catalysis using metal ions with orthogonal properties results in a synergistic enhancement in reaction kinetics.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41121199
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