A Reader-Based Assay for m⁶A Writers and Erasers

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.