A Reader-Based Assay for m6A Writers and Erasers

Anal Chem. 2019 Feb 19;91(4):3078-3084. doi: 10.1021/acs.analchem.8b05500. Epub 2019 Feb 4.

Abstract

We have developed a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay to measure the catalytic activity of N6-methyladenosine (m6A) methyltransferases and demethylases. The assay detects m6A modifications using the natural m6A-binding proteins (m6A readers). The reaction product or substrate m6A-containing RNA and the m6A reader protein are fluorescently labeled such that their proximity during binding initiates Förster resonance energy transfer (FRET). We show that our HTRF assay can be used for high-throughput screening, which will facilitate the discovery of small-molecule modulators of m6A (de)methylases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AlkB Homolog 5, RNA Demethylase / analysis*
  • AlkB Homolog 5, RNA Demethylase / metabolism
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO / analysis*
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO / metabolism
  • Fluorescence Resonance Energy Transfer*
  • Humans
  • Methyltransferases / analysis*
  • Methyltransferases / metabolism

Substances

  • ALKBH5 protein, human
  • AlkB Homolog 5, RNA Demethylase
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO
  • FTO protein, human
  • METTL4 protein, human
  • Methyltransferases
  • METTL3 protein, human