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(2)(6)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 multi-color fluorescence imaging.