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 N 6 -methyladenosine, a prevalent mRNA base modification. Methylated Broccoli is nonfluorescent, but upon demethylation by the RNA demethylases Fat mass and obesityassociated protein (FTO) or ALKBH5 binds and activates the fluorescence of its cognate fluorophore. We describe a high-throughput

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... en 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. Graphical Abstract *