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A general strategy exploiting m5C duplex-remodelling effect for selective detection of RNA and DNA m5C methyltransferase activity in cells.


ABSTRACT: RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-pucker switch in CpG RNA duplex but triggers a B-to-Z transformation in CpG DNA duplex. Inspired by these different 'structural signatures', we developed a m5C-sensitive probe which fluoresces spontaneously in response to m5C-induced sugar-pucker switch, hence useful for sensing RNA:m5C methyltransferase activity. Through the use of this probe, we achieved real-time imaging and flow cytometry analysis of NOP2/Sun RNA methyltransferase 2 (NSUN2) activity in HeLa cells. We further applied the probe to the cell-based screening of NSUN2 inhibitors. The developed strategy could also be adapted for the detection of DNA:m5C methyltransferases. This was demonstrated by the development of DNA m5C-probe which permits the screening of DNA methyltransferase 3A inhibitors. To our knowledge, this study represents not only the first examples of m5C-responsive probes, but also a new strategy for discriminating RNA and DNA m5C methyltransferase activity in cells.

SUBMITTER: Yang T 

PROVIDER: S-EPMC7145549 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A general strategy exploiting m5C duplex-remodelling effect for selective detection of RNA and DNA m5C methyltransferase activity in cells.

Yang Tianming T   Low Joanne J A JJA   Woon Esther C Y ECY  

Nucleic acids research 20200101 1


RNA:5-methylcytosine (m5C) methyltransferases are currently the focus of intense research following a series of high-profile reports documenting their physiological links to several diseases. However, no methods exist which permit the specific analysis of RNA:m5C methyltransferases in cells. Herein, we described how a combination of biophysical studies led us to identify distinct duplex-remodelling effects of m5C on RNA and DNA duplexes. Specifically, m5C induces a C3'-endo to C2'-endo sugar-puc  ...[more]

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