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A Reader-Based Assay for m⁶A Writers and Erasers
- Wiedmer, Lars, Eberle, Stefanie Alexandra, Bedi, Rajiv Kumar, Śledź, Paweł, Caflisch, Amedeo
- Analytical chemistry 2019 v.91 no.4 pp. 3078-3084
- RNA, catalytic activity, energy transfer, fluorescence, methyltransferases, proteins, screening
- 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.