Identification of Specific N6-Methyladenosine RNA Demethylase FTO Inhibitors by Single-Quantum-Dot-Based FRET Nanosensors.
Anal Chem
; 92(20): 13936-13944, 2020 10 20.
Article
em En
| MEDLINE
| ID: mdl-32972135
ABSTRACT
The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of N6-methyladenosine (m6A) residues in mRNA, regulates the cellular level of m6A 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 m6A 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 m6A-containing substrate.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Transferência Ressonante de Energia de Fluorescência
/
Pontos Quânticos
/
Inibidores Enzimáticos
/
Dioxigenase FTO Dependente de alfa-Cetoglutarato
Tipo de estudo:
Diagnostic_studies
Limite:
Humans
Idioma:
En
Revista:
Anal Chem
Ano de publicação:
2020
Tipo de documento:
Article