Harnessing Methyltransferase‐Guided Targeting for Sequence‐Specific Proximity Labeling of DNA

ABSTRACT 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.