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.
BT - Analytical Chemistry C1 - 0244 DA - 2019 Feb 19 DO - 10.1021/acs.analchem.8b05500 IS - 4 J2 - Anal. Chem. LA - eng M3 - Research Article N2 -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.
PY - 2019 SP - 3078 EP - 3084 T2 - Analytical Chemistry TI - A reader-based assay for m6A writers and erasers VL - 91 SN - 1520-6882 ER -