Detecting RNA base methylations in single cells by in situ hybridization

Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~10(4)–10(7) cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m(6)(2)A, m(1)G and m(3)U) that destabilize Watson–Crick base pairs. Our method—methylation-sensitive RNA fluorescence in situ hybridization—detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.