Detecting RNA base methylations in single cells by in situ hybridization

Rohan T. Ranasinghe (), Martin R. Challand (), Kristina A. Ganzinger, Benjamin W. Lewis, Charlotte Softley, Wolfgang H. Schmied, Mathew H. Horrocks, Nadia Shivji, Jason W. Chin, James Spencer and David Klenerman
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Rohan T. Ranasinghe: University of Cambridge
Martin R. Challand: University of Bristol
Kristina A. Ganzinger: University of Cambridge
Benjamin W. Lewis: University of Cambridge
Charlotte Softley: University of Cambridge
Wolfgang H. Schmied: Medical Research Council Laboratory of Molecular Biology
Mathew H. Horrocks: University of Cambridge
Nadia Shivji: University of Cambridge
Jason W. Chin: University of Cambridge
James Spencer: University of Bristol
David Klenerman: University of Cambridge

Nature Communications, 2018, vol. 9, issue 1, 1-10

Abstract: Abstract 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 ~104–107 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 (m62A, m1G and m3U) 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.

Date: 2018
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DOI: 10.1038/s41467-017-02714-7

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