Fat mass and obesity-associated enzyme (FTO) can dynamically regulate N⁶-methyladenosine modification, and it is engaged in various cellular functions. Herein, we demonstrate the RNA demethylation-driven functional supramolecular structure for label-free detection of m⁶A modification eraser FTO in human breast tissues. The presence of FTO catalyzes the removal of methyl group in m⁶A, causing the cleavage of demethylated DNA by DpnII and the release of DNA primer. The resultant DNA primer hybridizes with circular template to initiate isothermal rolling circle amplification (RCA), producing abundant long ssDNA polymers with repeating sequences of G-quadruplex. Subsequently, N-methylmesoporphyrin IX (NMM) is selectively embedded into G-quadruplex DNAzyme to form a supramolecular NMM-G-quadruplex structure for the generation of an amplified fluorescence signal. Benefiting from high selectivity of DpnII toward demethylated DNA, high amplification efficiency of RCA, and high signal-to-noise ratio of G-quadruplex-NMM system, this assay can sensitively detect FTO with a limit of detection (LOD) of 3.10 × 10⁻¹⁶ M, screen RNA demethylase inhibitors, quantify FTO activity in cancer cells, and discriminate FTO activity between breast cancer patient tissues and healthy person tissues. Importantly, this assay can be homogeneously conducted in a label-free manner, with great potential in RNA demethylases-related pathogenesis research and clinical diagnostics.

脂肪量和肥胖相关酶（FTO）可以动态调节N ⁶ -甲基腺苷修饰，参与多种细胞功能。在此，我们展示了 RNA 去甲基化驱动的功能性超分子结构，用于无标记检测人乳腺组织中的 m ⁶ A 修饰橡皮擦 FTO。^{FTO的存在催化m 6 A}中甲基的去除，引起Dpn II对去甲基化DNA的切割，释放DNA引物. 所得 DNA 引物与环状模板杂交以启动等温滚环扩增 (RCA)，产生大量具有 G-四链体重复序列的长 ssDNA 聚合物。随后，N-甲基中卟啉 IX (NMM) 被选择性地嵌入到 G-四链体 DNAzyme 中，形成超分子 NMM-G-四链体结构，用于产生放大的荧光信号。得益于Dpn的高选择性、RCA 的高扩增效率和 G-quadruplex-NMM 系统的高信噪比，该检测可以灵敏地检测 FTO，检测限 (LOD) 为 3.10 × 10⁻¹⁶ M，筛选 RNA 去甲基化酶抑制剂，量化癌细胞中的 FTO 活性，并区分乳腺癌患者组织和健康人组织之间的 FTO 活性。重要的是，这种测定可以以无标记的方式均匀进行，在 RNA 去甲基化酶相关的发病机制研究和临床诊断学中具有巨大潜力。