Harnessing Methyltransferase-Guided Targeting for Sequence-Specific Proximity Labeling of DNA.

Methyltransferase (MTase)-based DNA labeling has become a powerful strategy for genomic and epigenetic analysis because of its unique ability to recognize and functionalize DNA sequences in a site-specific manner. Expanding this toolbox is essential to fully exploit MTases as programmable molecular guides. Here, we introduce an MTase-directed proximity labeling approach that enables sequence-specific DNA modification beyond the natural catalytic transfer site: GLOW, Guided Labeling Outside the natural site With MTases. Using newly designed S-adenosyl-L-methionine (SAM) analogues, we demonstrate sequence-specific DNA labeling revealed by single-molecule fluorescence imaging and gel-based restriction enzyme assays, confirming that labeling occurs adjacent to, rather than within, the canonical recognition site. Unlike conventional MTase-mediated methods, this strategy provides enhanced ligand stability and avoids interference from endogenous DNA methylation, thereby broadening its potential to complex genomic contexts. These findings establish MTase-guided proximity labeling as a conceptually new mode of enzymatic targeting that enriches the chemical biology toolkit for sequence-specific DNA modification.