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A reader-based assay for m6A writers and erasers

L. Wiedmer; S.A. Eberle; R.K. Bedi; P. Śledź; A. Caflisch

Journal: Anal. Chem.
Year: 2019
Volume: 91
Issue: 4
Pages: 3078-3084
DOI: 10.1021/acs.analchem.8b05500
Type of Publication: Journal Article

DNA methylation; enzyme activity; Enzymes; epitranscriptomics; Fluorescence Resonance Energy Transfer; FRET; High-Throughput Screening Assays; m6A


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.