3.5. Enzyme Inhibition Assay

The enzymatic assay was modified from Huang et al. [29] The experiments were conducted in reaction buffer (50 mM Tris-HCl, pH 7.5, 300 μM 2OG, 280 μM (NH₄)₂Fe(SO₄)₂, and 2 mM L-ascorbic acid). The reaction mixture contained 200 ng methylated N⁶-adenine RNA probe (SEQ ID NO: 1) (5′-CUUGUCAm6ACAGCAGA-3′, Dharmacon, Lafayette, CO, USA) and 10 nM FTO protein and different concentrations of ligands (1 nM to 100 µM). Reactions were incubated on 96-well plate for 2 h at RT. After that, the amount of m⁶A that was measured using EpiQuik m⁶A RNA methylation Quantification Colorimetric Kit (Epigentek, Farmingdale, NY, USA).

The inhibitory effect IE of compounds on RNA probe demethylation by FTO was calculated as the enhancement of the m⁶A amount as compared to the negative control (DMSO) relative to the difference between m⁶A amounts of the positive control (max inhibition) and the negative control (Equation (2))

where C_inh, C_inh(max), and C_DMSO are the amounts of m⁶A at a given concentration of the inhibitor, maximum inhibition and in the case of DMSO, respectively.