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; David Klenerman

doi:10.1038/s41467-017-02714-7

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, David Klenerman

^*Corresponding author for this work

Research output: Contribution to journal › Article (Academic Journal) › peer-review

29 Citations (Scopus)

430 Downloads (Pure)

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 (m⁶ ₂A, 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.

Original language	English
Article number	655
Number of pages	10
Journal	Nature Communications
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41467-017-02714-7
Publication status	Published - 13 Mar 2018

Access to Document

10.1038/s41467-017-02714-7

Full-text PDF (final published version)
This is the final published version of the article (version of record). It first appeared online via Nature at https://www.nature.com/articles/s41467-017-02714-7 . Please refer to any applicable terms of use of the publisher.
Final published version, 1.44 MBLicence: CC BY
Supplementary figure PDF
This is the final published version of the article (version of record). It first appeared online via Nature at https://www.nature.com/articles/s41467-017-02714-7 . Please refer to any applicable terms of use of the publisher.
Final published version, 6.44 MBLicence: CC BY

Handle.net

Persistent link

Cite this

@article{c62f9f4b4b7b4f6386d379a57bdb0ee3,

title = "Detecting RNA base methylations in single cells by in situ hybridization",

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 (m6 2A, 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.",

author = "Ranasinghe, {Rohan T.} and Challand, {Martin R.} and Ganzinger, {Kristina A.} and Lewis, {Benjamin W.} and Charlotte Softley and Schmied, {Wolfgang H.} and Horrocks, {Mathew H.} and Nadia Shivji and Chin, {Jason W.} and James Spencer and David Klenerman",

year = "2018",

month = mar,

day = "13",

doi = "10.1038/s41467-017-02714-7",

language = "English",

volume = "9",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Springer Nature",

number = "1",

}

TY - JOUR

T1 - Detecting RNA base methylations in single cells by in situ hybridization

AU - Ranasinghe, Rohan T.

AU - Challand, Martin R.

AU - Ganzinger, Kristina A.

AU - Lewis, Benjamin W.

AU - Softley, Charlotte

AU - Schmied, Wolfgang H.

AU - Horrocks, Mathew H.

AU - Shivji, Nadia

AU - Chin, Jason W.

AU - Spencer, James

AU - Klenerman, David

PY - 2018/3/13

Y1 - 2018/3/13

N2 - 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 (m6 2A, 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.

AB - 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 (m6 2A, 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.

UR - http://www.scopus.com/inward/record.url?scp=85042022686&partnerID=8YFLogxK

U2 - 10.1038/s41467-017-02714-7

DO - 10.1038/s41467-017-02714-7

M3 - Article (Academic Journal)

C2 - 29440632

SN - 2041-1723

VL - 9

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 655

ER -

Detecting RNA base methylations in single cells by in situ hybridization

Abstract

Access to Document

Handle.net

Other files and links

Embargoed Document

Fingerprint

Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases

Professor Jim Spencer

Cite this

Detecting RNA base methylations in single cells by in situ hybridization

Abstract

Access to Document

Handle.net

Other files and links

Embargoed Document

Fingerprint

Projects

Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases

Profiles

Professor Jim Spencer

Cite this