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Related Experiment Video

Updated: Nov 25, 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

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

Yuru Wang^1,2, Christopher D Katanski¹, Christopher Watkins¹

¹Department of Biochemistry and Molecular Biology, USA.

Nucleic Acids Research

|December 18, 2020

Summary

This summary is machine-generated.

Researchers developed a high-throughput screening method to discover AlkB enzyme mutants with enhanced DNA/RNA demethylation activity. This new tool aids in identifying modified nucleic acids and evolving repair enzymes.

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

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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

Published on: August 8, 2016

Area of Science:

Biochemistry
Molecular Biology
Enzymology

Background:

AlkB is a DNA/RNA repair enzyme crucial for removing alkylation damage.
AlkB is a valuable tool for tRNA sequencing and identifying mRNA modifications.
Current methods for identifying improved AlkB mutants rely on targeted mutagenesis.

Purpose of the Study:

To develop a high-throughput screening method for evaluating AlkB variants.
To identify novel AlkB mutants with enhanced demethylation activity on RNA and DNA.
To enable the in vitro evolution of demethylase enzymes.

Main Methods:

A fluorogenic RNA aptamer assay was designed to detect demethylation activity.
Modified RNA/DNA residues in the aptamer block reverse transcription or cause fluorescence loss.
Demethylation by AlkB variants restores fluorescence, enabling high-throughput screening.

Main Results:

A novel AlkB variant with improved activity against N1-methylguanosine (m1G) in RNA was identified.
Candidate AlkB variants with demethylating activity on O6-methylguanosine (O6mG) DNA substrates were discovered.
The screening method successfully identified variants with enhanced enzymatic function.

Conclusions:

A robust high-throughput screening method for demethylase evolution has been established.
This method facilitates the discovery of AlkB variants with improved RNA and DNA repair capabilities.
The developed screening platform is applicable to the in vitro evolution of various demethylase enzymes.