| Assay Method Information | |
| | FTO inhibition Assay (Enzymatic Inhibition) |
| Description: | The inhibition assay was adapted from a publication about Maz-F coupled FTO demethylase activity assay (Chem Commun. 2017 Nov. 30; 53(96):12930-12933. doi: 10.1039/c7cc07699a). FTO-catalyzed demethylation activity was measured in a 10 μl reaction system containing 20 mM HEPES buffer (pH 7.5), 20 μM a-KG, 10 M (NH4)2Fe(SO4)2, 1.5 mM L-ascorbic acid, 2 μM DRNA with m6A (5′-FAM-d(CAT)r(GG-m6A-CA)d(TATGT)-BHQ1-3′), 0.15 μM FTO protein. The reaction system was incubated at 32° C. for 45 min. Aliquot MazF into each well above (40 ul 1×Maz-F+10 ul the reaction/well), the final concentration is 50 nM, and the reaction system was incubated at 37° C. for 25 min. MazF cleaves only nonmethylated DRNA (-ACA-), permitting specific detection of the methylation states of DRNA. Maz-F readily cleaves the DRNA and eventually liberates 5′-FAM, which gives a strong fluorescence signal around 535 nm (485 nm excitation-535 emission). |
| Affinity data for this assay | |
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