脂肪质量与肥胖相关蛋白（FTO）在N6-甲基腺苷（m6A）表观遗传修饰的可逆调控中起着重要作用，并且FTO的过度表达与多种人类疾病（例如肥胖和癌症）的发生密切相关。在这篇论文中，我们展示了利用单碱基扩展和连接驱动的多个DNA酶的构建，用于单分子检测肿瘤组织中的FTO。当目标FTO存在时，m6A-RNA会被特异性去甲基化，并随后作为引物与锁链探针结合，启动单碱基扩展和连接反应，生成闭合模板探针。添加phi29 DNA聚合酶后，开始滚圈扩增（RCA）反应，产生大量依赖于Mg2+的DNA酶重复序列。随后，DNA酶循环消化信号探针，释放大量可以通过单分子成像精确计数的Cy5分子。利用聚合酶/连接酶介导的缺口填充和连接的序列特异性，以及RCA的高扩增效率，该生物传感器具有优异的特异性和高灵敏度，检测限为5.96 × 10-16 M。它可用于筛选FTO抑制剂和定量单细胞水平的FTO活性。此外，所提出的策略可以准确区分健康个体和乳腺癌患者组织中的FTO表达水平，为药物发现、m6A修饰相关研究和临床诊断提供了一个新的平台。

Fat mass and obesity-associated proteins (FTO) play an essential role in the reversible regulation of N6-methyladenosine (m6A) epigenetic modification, and the overexpression of FTO is closely associated with the occurrence of diverse human diseases (e.g., obesity and cancers). Herein, we demonstrate the construction of multiple DNAzymes driven by single base elongation and ligation for the single-molecule monitoring of FTO in cancer tissues. When target FTO is present, the m6A-RNA is specifically demethylated and subsequently acts as a primer to combine with the padlock probe, initiating single-base elongation and ligation reaction to generate a closed template probe. Upon the addition of phi29 DNA polymerase, a rolling circle amplification (RCA) reaction is initiated to produce large numbers of Mg2+-dependent DNAzyme repeats. Subsequently, the DNAzymes cyclically digest the signal probes, liberating numerous Cy5 molecules that can be precisely counted by single-molecule imaging. Taking advantage of the sequence specificity of the polymerase/ligase-mediated gap-filling and ligation as well as the high amplification efficiency of RCA, this biosensor shows excellent specificity and high sensitivity with a detection limit of 5.96 × 10-16 M. It can be applied to screen FTO inhibitors and quantify FTO activity at the single-cell level. Moreover, the proposed strategy can accurately distinguish the FTO expression level in tissues of healthy individuals and breast cancer patients, providing a new platform for drug discovery, m6A modification-related research, and clinical diagnostics.