Harnessing S. aureus tRNA Guanine Transglycosylase for Efficient Site‐Specific Covalent Labeling of DNA

Site-specific DNA modification enables the construction of functional nucleic acid architectures for imaging, diagnostics, and information encoding. Bacterial tRNA guanine transglycosylases (TGTs) have been shown to exchange guanine-34 in cognate tRNAs for prequeuosine1 (preQ1) analogs. Escherichia coli TGT (EcTGT) has been widely harnessed for site-specific RNA labeling. However, EcTGT exhibits slow labeling kinetics and lower efficiency on DNA. Here, we demonstrate that TGT from Staphylococcus aureus (SaTGT) is a superior enzyme for DNA modification. Like EcTGT, SaTGT recognizes only the anticodon stem-loop and efficiently modifies the DNA hairpin dECYMH, whereas EcTGT shows no appreciable modification. Using the S. aureus tRNAHis-based hairpin as a starting point, we identified the most promising scaffolds. Rational loop optimization yielded dSAH-6, which is labeled by SaTGT with near-quantitative conversion under standard conditions. Side-by-side comparisons revealed that SaTGT outperforms EcTGT in both yield and kinetics across multiple preQ1 derivatives and hairpin sequences. Importantly, the optimized dSAH-6 sequence enables robust labeling at 5'-, 3'-, or internal positions in longer ssDNA constructs and supports efficient multi-site incorporation. These findings significantly expand the DNA-TAG toolbox and highlight how natural variation in TGT substrate promiscuity can be exploited for improved nucleic acid modification technologies.