Simultaneous In Situ Detection of m⁶A-Modified and Unmodified RNAs Using DART-FISH

N⁶-methyladenosine (m⁶A) is an abundant mRNA modification which plays important roles in regulating RNA function and gene expression. Traditional methods for visualizing mRNAs within cells cannot distinguish m⁶A-modified and unmodified versions of the target transcript, thus limiting our understanding of how and where methylated transcripts are localized within cells. Here, we describe DART-FISH, a visualization technique which enables simultaneous detection of both m⁶A-modified and unmodified target transcripts. DART-FISH combines m⁶A-dependent C-to-U editing with mutation-selective fluorescence in situ hybridization to specifically detect methylated and unmethylated transcript copies, enabling the investigation of m⁶A stoichiometry and methylated mRNA localization in single cells.