Multiplexed In Situ\nImaging of Site-Specific m6A Methylation\nwith Proximity Hybridization Followed by Primer Exchange Amplification\n(m6A-PHPEA)

Post-transcriptional modification of N6-methyladenosine\n(m6A) is\ncrucial for ribonucleic acid (RNA) metabolism and cellular function.\nThe ability to visualize site-specific m6A methylation at the single-cell\nlevel would markedly enhance our understanding of its pivotal regulatory\nfunctions in the field of epitranscriptomics. Despite this, current\nin situ imaging techniques for site-specific m6A are constrained,\nposing a significant barrier to epitranscriptomic studies and pathological\ndiagnostics. Capitalizing on the precise targeting capability of deoxyribonucleic\nacid (DNA) hybridization and the high specificity of the m6A antibody,\nwe present a method, termed proximity hybridization followed by primer\nexchange amplification (m6A-PHPEA), for the site-specific imaging\nof m6A methylation within cells. This approach enables high-resolution,\nsingle-cell imaging of m6A methylation across various RNA molecules\ncoupled with efficient signal amplification. We successfully imaged\nthree distinct m6A methylation sites concurrently in multiple cell\ntypes, revealing cell-to-cell variability in expression levels. This\nmethod promises to illuminate the dynamics of m6A-modified RNAs, potentially\nrevolutionizing epitranscriptomic research and the development of\nadvanced pathological diagnosis for chemical modifications.