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154018 https://hdl.handle.net/2286/R.I.35429 http://rightsstatements.org/vocab/InC/1.0/ All Rights Reserved 2015 ix, 113 pages : illustrations (some color) Doctoral Dissertation Academic theses Text eng Dunn, Matthew Ryan Chaput, John C LaBaer, Joshua Lake, Douglas Mangone, Marco Arizona State University Partial requirement for: Ph.D., Arizona State University, 2015 Includes bibliographical references (pages 99-113) Field of study: Molecular and cellular biology Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine. Molecular Biology Biochemistry Polymerase Engineering synthetic genetics Threose Nucleic Acid Xenonucleic Acids Polymers Synthetic Biology DNA polymerases RNA polymerases Nucleic Acids Developing engineered polymerases for practical applications in synthetic biology