PubAg

Main content area

A Reader-Based Assay for m⁶A Writers and Erasers

Author:
Wiedmer, Lars, Eberle, Stefanie Alexandra, Bedi, Rajiv Kumar, Śledź, Paweł, Caflisch, Amedeo
Source:
Analytical chemistry 2019 v.91 no.4 pp. 3078-3084
ISSN:
1520-6882
Subject:
RNA, catalytic activity, energy transfer, fluorescence, methyltransferases, proteins, screening
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.
Agid:
6311726