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Related Experiment Video

Updated: Jul 27, 2025

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

Published on: August 5, 2022

Blocker-dUThiophene poly tailing-based method for assessing methyl transferase activity.

Kazi Morshed Alom¹, Young Jun Seo²

¹Department of Chemistry, Jeonbuk National University, Jeonju, 561-756, Republic of Korea.

Analytical and Bioanalytical Chemistry

|June 8, 2023

Summary

We developed a novel assay for detecting methyl transferase (MTase) activity. This method uses a specially designed DNA probe and enzymatic reactions to generate a fluorescent signal, enabling sensitive MTase detection.

Keywords:

DNA methylation Diagnostics Fluorescence Methyltransferase S-Adenosyl methionine

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Last Updated: Jul 27, 2025

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

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Published on: August 5, 2022

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Published on: August 7, 2021

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

Methyl transferases (MTases) are crucial enzymes involved in various biological processes.
Accurate detection of MTase activity is essential for understanding cellular functions and disease mechanisms.
Existing methods for MTase detection can lack sensitivity or specificity.

Purpose of the Study:

To develop a novel, selective, and sensitive method for detecting methyl transferase activity.
To establish a fluorescence-based assay for MTase detection using a unique dsDNA probe.
To characterize the performance of the developed assay, including its limit of detection and selectivity.

Main Methods:

A dsDNA probe with C3 spacers and a methyl transferase recognition sequence was designed.
Methylation by MTase unblocks the probe, allowing cleavage by DpnI endonuclease.
TdT polymerase-mediated poly-tailing with fluorescent dUThioTP generates a detectable signal.

Main Results:

The method demonstrates selective cleavage of methylated dsDNA probes.
A limit of detection of 0.049 U/mL was achieved, indicating high sensitivity.
The assay showed good selectivity for MTase activity, with no fluorescence in the absence of MTase.

Conclusions:

The developed method provides a sensitive and selective approach for detecting methyl transferase activity.
This assay has potential for accurate analysis of MTase activity in various biological samples.
The fluorescence-based detection offers a robust platform for MTase research.