Single-residue engineering of Klenow fragment enables broad acceptance of chemically modified nucleotides.

Xeno nucleic acids (XNAs) exhibit enhanced chemical stability and significant resistance to nuclease degradation, making them attractive for synthetic biology and therapeutic development. Most engineered XNA polymerases are derived from thermophilic organisms and exhibit limited catalytic activity under physiological conditions, thereby limiting their broader application. We report a single-residue mutant (F762A) of the mesophilic Family A DNA polymerase I Klenow fragment that synthesizes DNA, RNA, 2'-F-RNA, and FANA with yields exceeding 85% at 37°C, as well as 2'-OMe-RNA, HNA, phosphorothioate-, and (methyl) pseudoU-containing oligonucleotides, confirming its broad substrate compatibility. Compared to wild-type, F762A exhibits up to 90-fold higher catalytic efficiency for modified nucleotides while maintaining an overall error rate below 1.19 × 10-3. F762A functions efficiently under physiological metal ion concentrations and molecular crowding, with reverse transcriptase activity and 2'-F-RNA-templated self-replication. Unlike thermophilic XNA polymerases, nearly inactive at 37°C, F762A also extends DNA and RNA primers to generate chimeric XNAs. Molecular dynamics simulations show F762A relieves steric hindrance from the phenylalanine side chain, improving modified nucleotide accommodation while maintaining polymerase structural integrity. These findings establish a foundation for polymerase-mediated XNA synthesis under physiological conditions and expand the potential of XNAs in synthetic biology and biotechnology.