UC Irvine

Modern chemical biology seeks genetic polymers with enhanced pharmacological and physicochemical properties for applications in therapeutics, diagnostics, and biotechnology. Reprogramming DNA polymerases offers a controlled, scalable, and surprisingly versatile method of synthesis, paving the way for the selection of functional polymers. However, the precision that makes DNA polymerases finely tuned, fast, and faithful molecular machines also limits their ability to accept noncognate substrates. This thesis examines the adaptability of hyperthermophilic archaeal B-family DNA polymerases toward polymers with unique benefits and challenges: phosphonomethylthreosyl nucleic acid (pTNA), threose nucleic acid (TNA), and RNA.pTNA is an orthogonal polymer intended to remain insulated from cellular DNA and RNA, raising fundamental questions about how it can be copied from a DNA template. Crystallographic analysis showed that an engineered DNA polymerase can organize canonical Watson-Crick base pairing between nascent pTNA and templating DNA, stabilizing an otherwise unfavorable DNA– pTNA heteroduplex.TNA, by contrast, can exchange genetic information with DNA and resists nuclease degradation, making it valuable for molecular evolution and biotechnology. By combining homologous recombination and random mutagenesis with high-throughput microfluidic screening, this work reports the directed evolution of TNA polymerase 10-92, which synthesizes TNA at ~1nt s-1 with >99% fidelity. Its utility is demonstrated by efficient synthesis of TNA aptamers, including molecules containing C5-modified uracil residues.RNA is a highly versatile natural genetic polymer, and its synthesis can benefit from a thermostable, primer-dependent polymerase. Homologous recombination and microfluidic screening yielded RNA polymerase C28, which operates at ~3 nt s-1 with >99% fidelity and supports long-range RNA synthesis, reverse transcription, chimeric DNA–RNA PCR amplification, and incorporation of several 2′-fluoro and base-modified ribonucleotides.Structures of 10-92 and its evolutionary intermediates revealed extensive, long-range remodeling that progressively refined the active site to accommodate TNA. Fidelity and catalytic rate followed distinct evolutionary trajectories, demonstrating that these properties can be improved through separable mechanisms. Mutational analysis further suggested that a highly stable scaffold permitted the accumulation of numerous substitutions predicted to be stabilityneutral, whereas a smaller set of distal mutations, predicted to be destabilizing, fine-tuned catalysis by altering flexibility and rigidity. Structural analysis of C28 also shows that efficient RNA synthesis arose from steric-gate removal and altered electrostatics, and likely from distributed interactions that remodeled substrate recognition.Together, this work establishes 10-92 and C28 as practical tools for synthetic genetics and reveals how polymerases can stabilize otherwise unfavorable base pairing, reshape catalytic geometry through distant sequence changes, and enhance fidelity through mechanisms separable from catalytic rate.