Developing Engineered Polymerases for Practical Applications in Synthetic Biology

• Other Authors: Dunn, Matthew Ryan (Author)
• Format: Doctoral Thesis
• Language: English
• Published: 2015
• Subjects: Molecular biology; Biochemistry; Polymerase Engineering; Synthetic Genetics; Threose Nucleic Acid; Xenonucleic Acids
• Online Access: http://hdl.handle.net/2286/R.I.35429

abstract: 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. === Dissertation/Thesis === Doctoral Dissertation Molecular and Cellular Biology 2015