Detecting RNA base methylations in single cells by in situ hybridization

  • Nat Commun. 2018 Feb 13;9(1):655. doi: 10.1038/s41467-017-02714-7.
Rohan T Ranasinghe  1 ,  Martin R Challand  2  3 ,  Kristina A Ganzinger  4  5 ,  Benjamin W Lewis  4 ,  Charlotte Softley  4 ,  Wolfgang H Schmied  6 ,  Mathew H Horrocks  4 ,  Nadia Shivji  4 ,  Jason W Chin  4  6 ,  James Spencer  7 ,  David Klenerman  4
Affiliations
  • 1. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. [email protected].
  • 2. School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TD, UK. [email protected].
  • 3. School of Biochemistry, University of Bristol, Bristol, BS8 1TD, UK. [email protected].
  • 4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
  • 5. Max-Planck-Institut für Biochemie (MPI for Biochemistry), 82152, Martinsried, Germany.
  • 6. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK.
  • 7. School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TD, UK.
PMID: 29440632 DOI: 10.1038/s41467-017-02714-7
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.

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