All known living cells contain a complex set of molecular machinery to support their growth and replication. However, the earliest cells must have been much simpler, consisting of a compartment and a genetic material to allow for Darwinian evolution. To study these intermediates, plausible model `protocells' must be synthesized in the laboratory since no fossils remain. Recent work has shown that fatty acids can self-assemble into vesicles that are able to grow and divide through simple mechanisms. However, a self-replicating protocell genome has not yet been developed. Here we discuss studies of systems that allow for the copying of nucleic acid templates without enzymes and how they could be developed into a genetic material. The simplest method of non-enzymatic template copying is to use activated nucleotides that can polymerize spontaneously. We used the template-directed addition of 2-methylimidazole-activated guanylate to an RNA primer as a model system to study the mechanism of polymerization. We found that general acid-base catalysis is not involved and the reaction has a maximum rate at pH 7.5. Inner shell contacts to Mg2+ at the reaction center are likely important to orient the 3'-hydroxyl and phosphate for inline attack. Furthermore, we found that there is likely an interaction between activating groups of adjacent monomers, which has important implications for activating group optimization and in situ monomer activation. We then report the synthesis and non-enzymatic polymerization of the non-natural 2'-amino-threose nucleic acids (2'-NH2-TNA). We found that 2'-NH2-TNA nucleotides polymerized more slowly than similar amino-substituted ribonucleic acids. Although single-stranded TNA is less flexible than RNA, we found that polymerization of 2'-NH2-TNA nucleotides was slower on TNA templates. These results suggest that TNA would have been a poor competitor to RNA in the origin of life. Finally, we report the development of initiator nucleotides that use `click' chemistry to site-specifically label RNA transcripts. We propose a scheme for an in vitro selection experiment using these nucleotides to isolate a ribozyme that could catalyze RNA replication. Altogether, these results will help guide the development of a self-replicating nucleic acid system that can be integrated with a compartment to create a protocell.
|Advisor:||Szostak, Jack W.|
|Commitee:||Blower, Michael D., Liu, David R., McLaughlin, Larry W., Ruvkun, Gary, Seed, Brian, Walker, Suzanne|
|Department:||Biology: Medical Sciences, Division of|
|School Location:||United States -- Massachusetts|
|Source:||DAI-B 75/05(E), Dissertation Abstracts International|
|Subjects:||Genetics, Chemistry, Biochemistry|
|Keywords:||Click chemistry, Origin of life, Protocell, Self replication, Synthetic biology|
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