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A Reader-Based Assay for m6A Writers and Erasers.

Lars Wiedmer1, Stefanie Alexandra Eberle1, Rajiv Kumar Bedi1

  • 1Department of Biochemistry , University of Zurich , Winterthurerstrasse 190 , CH-8057 Zurich , Switzerland.

Analytical Chemistry
|February 5, 2019
PubMed
Summary
This summary is machine-generated.

We developed a new assay to measure the activity of N6-methyladenosine (m6A) enzymes. This method uses fluorescence to detect m6A modifications, aiding the discovery of new drugs.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Epigenetics

Background:

  • N6-methyladenosine (m6A) is a crucial epitranscriptomic modification.
  • m6A methyltransferases and demethylases regulate m6A levels.
  • Developing robust assays for these enzymes is vital for understanding their roles and for drug discovery.

Purpose of the Study:

  • To develop a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay.
  • To measure the catalytic activity of m6A methyltransferases and demethylases.
  • To facilitate high-throughput screening for small-molecule modulators of m6A (de)methylases.

Main Methods:

  • Developed a homogeneous time-resolved fluorescence (HTRF) assay.
  • Utilized natural m6A-binding proteins (m6A readers) for detection.
  • Employed Förster resonance energy transfer (FRET) between fluorescently labeled RNA and reader proteins.

Main Results:

  • The HTRF assay successfully measures m6A methyltransferase and demethylase activity.
  • The assay detects m6A modifications using m6A reader proteins.
  • Demonstrated the assay's suitability for high-throughput screening.

Conclusions:

  • The developed HTRF assay is a sensitive and efficient tool for studying m6A (de)methylases.
  • This assay platform can accelerate the discovery of novel therapeutics targeting m6A pathways.
  • Facilitates the identification of small-molecule modulators for epigenetic drug development.