Department of Biochemistry , University of Zurich , Winterthurerstrasse 190 , CH-8057 Zurich , Switzerland.
Anal Chem. 2019 Feb 19;91(4):3078-3084. doi: 10.1021/acs.analchem.8b05500. Epub 2019 Feb 4.
We have developed a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay to measure the catalytic activity of N-methyladenosine (mA) methyltransferases and demethylases. The assay detects mA modifications using the natural mA-binding proteins (mA readers). The reaction product or substrate mA-containing RNA and the mA 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 mA (de)methylases.
我们开发了一种基于均相时间分辨荧光（HTRF）的酶测定法，用于测量 N6-甲基腺苷（m6A）甲基转移酶和去甲基酶的催化活性。该测定法使用天然的 m6A 结合蛋白（m6A 读码器）来检测 m6A 修饰。反应产物或底物含有 m6A 的 RNA 和 m6A 读码器蛋白被荧光标记，使得它们在结合时的接近度引发Förster 共振能量转移（FRET）。我们表明，我们的 HTRF 测定法可用于高通量筛选，这将有助于发现 m6A（去）甲基酶的小分子调节剂。
