Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes.
Fluorescent RNA Aptamers as a Tool to Study RNA-Modifying Enzymes.
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DOI：
10.1016/j.chembiol.2015.11.018
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发表时间：
2016-03-17
影响因子：
8.6
通讯作者：
Jaffrey SR
中科院分区：
文献类型：
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作者：
Svensen N;Jaffrey SR
RNA修饰酶由于缺乏荧光底物而难以测定。在这里，我们表明，花椰菜，以前报道的荧光RNA染料复合物，可以修改包含N6-甲基腺苷，一个普遍的mRNA碱基修饰。甲基化的西兰花是无荧光的，但在通过RNA去甲基化酶脂肪质量和肥胖相关蛋白（FTO）或ALKBH 5结合并激活其同源荧光团的荧光。我们描述了使用荧光甲基化西兰花底物HTS测定法进行的FTO抑制剂的高通量筛选，在LOPAC 1280文库中Z '因子> 0.8。这允许鉴定新的高亲和力FTO抑制剂。这些化合物中的几种对FTO的选择性超过相关的脱甲基酶ALKBH 5，并增加细胞中内源性FTO靶mRNA的甲基化。最后，我们表明，西兰花可以修改包含其他碱基的修改，这表明这种方法可以普遍适用于测定不同的RNA修饰酶。
RNA-modifying enzymes are difficult to assay due to the absence of fluorometric substrates. Here we show that the Broccoli, a previously reported fluorescent RNA-dye complex, can be modified to contain N6-methyladenosine, a prevalent mRNA base modification. Methylated Broccoli is nonfluorescent, but upon demethylation by the RNA demethylases Fat mass and obesity-associated protein (FTO) or ALKBH5 binds and activates the fluorescence of its cognate fluorophore. We describe a high-throughput screen for FTO inhibitors using the fluorogenic methylated Broccoli substrate HTS assay performs robustly with a Z’-factor > 0.8 in the LOPAC1280 library. This allowed the identification of novel high-affinity FTO inhibitors. Several of these compounds were selective for FTO over the related demethylase, ALKBH5, and increase methylation of endogenous FTO target mRNAs in cells. Lastly, we show that Broccoli can be modified to contain other base modifications, suggesting that this approach could be generally applicable for assaying diverse RNA-modifying enzymes.