Identification of Specific N⁶-Methyladenosine RNA Demethylase FTO Inhibitors by Single-Quantum-Dot-Based FRET Nanosensors

Abstract

The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N 6 -methyladenosine (m 6 A) residues in mRNA, regulates the cellular level of m 6 A modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of m 6 A can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the m 6 A-containing substrate.

Label-free and sensitive detection of RNA demethylase FTO with primer generation rolling circle amplification

“…3D), respectively. The half maximal inhibition (IC 50 ) values are calculated to be 0.96 mM for diacerein and 30.91 mM for rhein, consistent with the values obtained by a single QD-based FRET sensor (1.51 mM) for diacerein 17 and a DNAzyme-based fluorescent assay (47.2 mM) for rhein, 24 suggesting the feasibility of this method for the screening of FTO inhibitors.…”

“…13 After the demethylation of ssDNA by FTO and ALKBH5, ssDNA is annealed with its complementary DNA sequence, followed by recognition and cleavage by Dpn II at the recognition site. 14 The integration of Dpn II with polyacrylamide gel electrophoresis (PAGE), 15 HPLC 16 and single-quantumdot (QD)-based Fo ¨rster resonance energy transfer (FRET) 17 makes the detection of FTO/ALKBH5 activity very simple. However, PAGE and HPLC usually involve large amounts of FTO/DNA substrates and complicated sample preparation steps; FRET assay requires careful calculation of the distance and angle between the donor and the acceptor for the achievement of high FRET efficiency.…”

“…In contrast, in the absence of FTO, the methylated RNA probe cannot be cleaved by MazF. 17 As a result, the PG-RCA reaction cannot be initiated and no fluorescence enhancement is observed.…”

“…Notably, the sensitivity of this method is 7 orders of magnitude higher than that of a methylation-switchable probe-based fluorescent assay (10 nM), 11 6 orders of magnitude higher than that of a hybridization chain reaction-based fluorescent assay (2.273 nM), 23 5 orders of magnitude higher than that of a DNAzyme-based fluorescent assay (0.5 nM), 24 and 1 order of magnitude higher than that of a single QD-based FRET sensor (7.9 Â 10 À14 M). 17 The improved sensitivity can be attributed to the following three factors: (1) the high specificity of the cleavage reaction catalyzed by MazF enables the discrimination of demethylated substrates from methylated substrates, (2) the modification of the 3 0 termini of the RNA probes by NH 2 efficiently prevents nonspecific amplification, (3) the introduction of NbÁBsmI makes the linear RCA reaction change to a PG-RCA reaction, significantly improving the amplification efficiency. Moreover, this method exhibits good selectivity towards FTO with no response to nonspecific enzymes/proteins (Fig.…”

Targeting the RNA demethylase FTO for cancer therapy

“…For example, nafamostat mesilate and clausine E bind and inhibit demethylase activity in vitro ; 92,93 MU06 was designed through a scaffold hopping approach, and further evaluation using molecular docking simulations indicated direct binding with FTO; 105 in a single-quantum-dot (QD)-based FRET nanosensor assay, diacerein, which has a similar structure to rhein, showed an inhibitory effect on FTO with an IC 50 of 1.5 μM. 75 …”