Single-cell Imaging of m6A modified RNA Using m6A-specific In Situ Hybridization mediated Proximity Ligation Assay (m6AISH-PLA)

N 6 -methyladenosine (m 6 A) modification - the most prevalent mammalian RNA internal modification - plays key regulatory roles in mRNA metabolism. Current approaches for m 6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m 6 A-specific in situ hybridization mediated proximity ligation assay (m 6 AISH-PLA) for cellular imaging of m 6 A RNA, allowing to identify m 6 A modification at specific location in RNAs and image m 6 A RNA with single-cell and single-molecule resolution. Using m 6 AISH-PLA, we investigated the m 6 A level and subcellular location of HSP70 RNA103-m 6 A in each cell in response to heat shock, and found an increased m 6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock stress. m 6 AISH-PLA can serve in the study of m 6 A RNA in single-cell for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.