UC Irvine

DNA polymerases are central to biology and biotechnology due to their ability to faithfully propagate genetic information. Expanding the substrate scope of these enzymes beyond natural nucleic acids represents a major challenge in enzyme engineering and is essential to the practical use of new classes of genetic polymers. This thesis describes the directed evolution, technological development, and structural analysis of polymerases capable of synthesizing α-L-threofuranosyl nucleic acid (TNA), a synthetic genetic polymer with a noncanonical sugar backbone that is resistant to nucleases and acid-mediated degradation.To overcome the inherent incompatibility between natural polymerases and TNA substrates, an integrated evolutionary strategy was employed. Starting from a homologous recombination library, iterative rounds of selection and random mutagenesis were used to traverse the polymerase fitness landscape and identify mutation combinations that collectively enable efficient TNA synthesis. This campaign yielded polymerase variant 10–92, which catalyzes TNA synthesis with near-natural performance, achieving catalytic rates of ~1 nt/s and fidelity > 99%. Structural analysis of the closed ternary complex revealed widespread structural remodeling to accommodate the altered geometry of TNA, highlighting the role of distributed mutations in enabling new enzymatic functions. Central to this effort was the development of a recombination-based diversification strategy, designed to interface with an established droplet-based microfluidic screening platform. This workflow enabled the generation of a highly diverse polymerase library and reliable recovery of active variants, supporting iterative rounds of evolution. These advances streamlined the enrichment and regeneration of functional variants, and established a general framework that can be adapted to a variety of DNA-modifying enzymes. Finally, by capturing and characterizing multiple evolutionary intermediates along the evolutionary trajectory of the 10-92 TNA polymerase, this thesis provides mechanistic insight into one example of how new enzymatic activities emerge. Structural and biochemical analyses reveal that improvements in catalytic efficiency and replication fidelity arise from distinct molecular bases, challenging the longstanding view that accuracy and catalysis are coupled events. Together, these findings establish recombination-guided evolution, high-throughput screening, and structural interrogation as powerful and complementary tools for expanding the enzymatic recognition of synthetic genetic polymers, with implications for synthetic biology, biotechnology, and the study of enzyme evolution.