N6甲基腺嘌呤酸脱甲基酶（m6A demethylase）催化去除m6A修饰以建立适当的RNA甲基化模式，其已被发现为有前景的疾病生物标志物和治疗靶点。已报道的m6A脱甲基酶测定常受困于繁琐的制剂、昂贵的试剂、放射性风险、有限的灵敏度和较差的特异性。在此我们开发了一种基于脱甲基引发指数信号放大的简单、选择性、无标记、高灵敏度的荧光生物传感器来进行m6A脱甲基酶测定。在该生物传感器中，m6A脱甲基酶催化的脱甲基反应可以保护环状DNA免受DpnI的消化，随后引发超支化滚动环形扩增，实现对ssDNA和dsDNA产物的指数信号放大。扩增的DNA信号可以通过SYBR Gold在无标记的方式下被敏感地和简单地检测到。该生物传感器避免了任何抗体、洗涤/分离步骤和荧光探针/猝灭器的使用，极大地简化了测定步骤并降低了测定成本。此外，该生物传感器具有良好的特异性和优异的灵敏度，检测限为1.2 fg/μL，优于传统的ELISA（36.3 pg/μL）。特别值得注意的是，该生物传感器能够在粗细胞提取物中直接监测m6A脱甲基酶活性，具有较高的准确性，并可进一步应用于m6A脱甲基酶抑制剂的筛选、不同细胞系中的m6A脱甲基酶活性测量以及在临床癌症和健康组织中区分m6A脱甲基酶水平，为RNA甲基化相关的生物医学研究、疾病诊断和药物发现提供了一种简便且可靠的平台。版权所有©2023，Elsevier B.V.出版。

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/μL, which is superior to conventional ELISA (36.3 pg/μL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.Copyright © 2023. Published by Elsevier B.V.