m7G-PIP: Spatially resolved live-cell mapping of m7G-centric RNA-protein interactomes

m⁷G modification regulates mRNA life cycle and drives progression of cancers and viral infections. Current detection methods, however, rely on antibodies and disrupt native cellular architecture, making live-cell in situ mapping of m⁷G an unmet challenge. To address this, we develop m⁷G-Proximity Interactome Profiling (m⁷G-PIP). This antibody-free technology uses proximity labeling to biotinylate biomolecules proximal to bound m⁷G sites, amplifying signals for nanoscale-resolution (< 20 nm) mapping of RNA-protein interactomes in live cells. Applying this technology to herpes simplex virus-1 (HSV-1), we reveal dynamic m⁷G-centered networks during infection and identify internal m⁷G modifications on viral transcripts—the first such evidence on HSV-1. The modular design of m⁷G-PIP is readily adaptable to profile other RNA modifications with binding proteins. Our work establishes a spatiotemporal framework for epitranscriptome regulation and provides a versatile platform for developing antivirals targeting RNA modification pathways.