FRET-based method for direct, real-time measurement of DNA methyltransferase activity

Yi Long; Krystian Ubych; Elodie Jagu; Robert K Neely

doi:10.1021/acs.bioconjchem.0c00612

FRET-based method for direct, real-time measurement of DNA methyltransferase activity

Yi Long, Krystian Ubych, Elodie Jagu, Robert K Neely

Chemistry

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Abstract

DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.

Original language	English
Pages (from-to)	192-198
Number of pages	7
Journal	Bioconjugate Chemistry
Volume	32
Issue number	1
Early online date	11 Dec 2020
DOIs	https://doi.org/10.1021/acs.bioconjchem.0c00612
Publication status	Published - 20 Jan 2021

Bibliographical note

Funding Information:
This work was supported by the Engineering and Physical Sciences Research Council (EPSRC, grant number EP/N020901/1).

Copyright:
© 2020 American Chemical Society

ASJC Scopus subject areas

Biotechnology
Bioengineering
Biomedical Engineering
Pharmacology
Pharmaceutical Science
Organic Chemistry

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1021/acs.bioconjchem.0c00612Licence: None: All rights reserved

LongY2021FRET
This document is the Accepted Manuscript version of a Published Work that appeared in final form in Bioconjugate Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see: https://doi.org/10.1021/acs.bioconjchem.0c00612
Accepted author manuscript, 819 KBLicence: None: All rights reserved

Cite this

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title = "FRET-based method for direct, real-time measurement of DNA methyltransferase activity",

abstract = "DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.",

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AU - Long, Yi

AU - Ubych, Krystian

AU - Jagu, Elodie

AU - Neely, Robert K

PY - 2021/1/20

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AB - DNA methyltransferase activity is associated with a host of diseases, including cancers, where global hypomethylation of the genome, as well as marked changes in local DNA methylation patterns, can be both diagnostic and prognostic for the disease. Despite this, we currently lack a method for directly measuring the activity of the DNA methyltransferases, which would support the development of DNA methyltransferase-targeted therapies. Here, we demonstrate an assay for the direct measurement of methyltransferase activity, in real time. We employ a fluorescent methyltransferase cofactor analogue, which when bound by the enzyme to a labeled target DNA sequence results in fluorescence resonance energy transfer (FRET) between the donor dye (DNA) and the acceptor dye (cofactor). We demonstrate that the method can be used to monitor the activity of DNA MTases in real time and can be applied to screen inhibitors of the DNA methyltransferases. We show this in both bulk phase and single molecule imaging experiments, highlighting the potential application of the assay in screening and biophysical studies of methyltransferase function.

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FRET-based method for direct, real-time measurement of DNA methyltransferase activity

Abstract

Bibliographical note

ASJC Scopus subject areas

UN SDGs

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