UC Irvine

Xeno-nucleic acids (XNAs) are artificial genetic polymers with unnatural sugar and backbone structures that confer unique physicochemical properties, such as enhanced biostability and improved RNA binding thermodynamics, making them attractive for a wide range of applications. One such XNA is threose nucleic acid (TNA), which features a threose sugar ring with a 2′-3′ phosphodiester backbone linkage that confers high nuclease resistance while still supporting anti-parallel Watson-Crick base-pairing with DNA and RNA. Initially investigated as a plausible RNA progenitor, TNA research has since expanded towards its development as a molecular tool. This dissertation examines TNA across three studies that progress from the characterization of one of TNA’s special properties to the application of TNA towards specific functions. First, to examine the mechanistic rationale behind the chemical resilience of TNA, the degradation kinetics of TNA under acidic, high-temperature conditions was assessed using RP-HPLC, mass spectrometry, and molecular dynamics simulations. TNA exhibited a half-life substantially longer than DNA and RNA, with strand cleavage occurring via β-elimination of the 2′-phosphodiester linkage, a mechanism distinct from natural nucleic acids and attributable to destabilization of the oxocarbenium intermediate responsible for depurination. Second, to demonstrate the utility of TNA in a therapeutic context, TNA was incorporated into the backbone architecture of an RNA-cleaving DNA enzyme (DNAzyme) through chemical evolution. The resulting TNA-modified lead construct exhibited enhanced catalytic performance and achieved allele-specific mRNA and protein knockdown of the oncogenic KRAS G12V mutation by evading RNase H1 activity in cells. Third, to explore the link between dynamism and evolvability in nucleic acid enzymes, RNA-cleaving TNA enzymes (threozymes) were evolved by in vitro selection and compared to the well-characterized 10-23 DNAzyme. Unlike the DNAzyme, which was most active at physiological temperature, the threozymes required elevated temperature to become catalytically active, a finding consistent with TNA's more conformationally restricted backbone that limits access to productive folding states and raises the energetic barrier to catalysis. Together, these findings establish TNA as a chemical tool for engineering nucleic acid enzymes and a model system for probing how backbone architecture governs catalytic evolvability in nucleic acids, carrying significant implications in the development of XNA-based molecular medicine and biotechnologies.