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

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Identification of Specific N6-Methyladenosine RNA Demethylase FTO Inhibitors by Single-Quantum-Dot-Based FRET Nanosensors
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-09-25 , DOI: 10.1021/acs.analchem.0c02828
Yan Zhang ₁ , Qing-Nan Li ₁ , Kaiyue Zhou ₂ , Qinfeng Xu ₂ , Chun-Yang Zhang ₁

Affiliation

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.

中文翻译：

基于单量子点的 FRET 纳米传感器鉴定特定的 N6-甲基腺苷 RNA 去甲基化酶 FTO 抑制剂

脂肪量和肥胖相关酶(FTO)可催化mRNA中N ⁶ -甲基腺苷(m ⁶ A)残基的去甲基化，调节m ⁶ A修饰的细胞水平，在人类肥胖和癌症中起关键作用。在此，我们开发了一种基于单量子点 (QD) 的荧光共振能量转移 (FRET) 传感器，用于识别特定的 FTO 去甲基化酶抑制剂。^{FTO介导的m 6}去甲基化A 可以诱导去甲基化 DNA 的切割以产生生物素化的 DNA 片段，这些片段可以作为捕获探针将 Cy5 标记的报告探针组装到 QD 表面，使 QD 和 Cy5 之间发生 FRET。抑制剂的存在可以抑制 FTO 去甲基化，从而消除 QD 和 Cy5 之间的 FRET。抑制剂对 FTO 去甲基化的抑制作用可以通过监测 Cy5 计数的减少来简单地评估。我们使用这种纳米传感器来筛选几种小分子抑制剂，并将双醋瑞因鉴定为 FTO 的高选择性抑制剂。Diacerein 可抑制 HeLa 细胞内源性 FTO 的去甲基化活性。有趣的是，双醋瑞因既不是 2-酮戊二酸 (2-OG) 的结构模拟物，也不是金属离子的螯合剂，^含6 A的底物。

更新日期：2020-10-21

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