N6-甲基腺苷(m6A)是真核生物信使RNA和长链非编码RNA中可逆的化学修饰。RNA甲基转移酶METTL3-METTL14复合物的异常表达可能会改变m6A甲基化水平，引起多种疾病，包括癌症。传统的METTL3-METTL14检测常常受到耗时的程序和灵敏度低的影响。在此，我们开发了一种基于MazF激活的末端脱氧核苷酸转移酶(TdT)辅助树枝状DNA结构组装的可控扩增机制，用于癌细胞和乳腺组织中METTL3-METTL14复合物活性的超灵敏检测。METTL3-METTL14复合物的存在催化了检测探针中m6A的形成，有效防止了MazF对甲基化探针的切割。带有3'-OH端的甲基化检测探针可以在磁珠上无模板聚合酶催化下作为引物进行聚合，产生长链聚胸腺嘧啶(poly-T)序列。然后，聚T序列与包含聚腺嘌呤(poly-A)序列的信号探针杂交，诱导TdT介导的聚合，随后与更多的聚A信号探针杂交，生成组装在磁珠上的树枝状DNA纳米结构。经过磁分离和高温处理后，信号探针从磁珠中解离，生成高荧光信号。该方法具有良好的特异性和高灵敏度，检测限为2.61×10-15 M，可以准确定量单个细胞中的METTL3-METTL14复合物。此外，它还可以筛选抑制剂，评估动力学参数，并区分正常组织和乳腺癌组织。版权所有©2023 Elsevier B.V.。保留所有权利。

N6-Methyladenosine (m6A) is a reversible chemical modification in eukaryotic messenger RNAs and long noncoding RNAs. The aberrant expression of RNA methyltransferase METTL3-METTL14 complex may change the m6A methylation level and cause multiple diseases including cancers. The conventional METTL3-METTL14 assays commonly suffer from time-consuming procedures and poor sensitivity. Herein, we develop a controllable amplification machinery based on MazF-activated terminal deoxynucleotidyl transferase (TdT)-assisted dendritic DNA structure assembly for ultrasensitive detection of METTL3-METTL14 complex activity in cancer cells and breast tissues. The presence of METTL3-METTL14 complex catalyzes the formation of m6A in detection probe, effectively preventing the cleavage of methylated detection probes by MazF. The methylated detection probes with 3'-OH termini can function as the primers for template-free polymerization catalyzed by TdT on magnetic beads (MBs), producing long chains of poly-thymidine (poly-T) sequences. Then poly-T sequences hybridize with signal probes that contain poly-adenine (poly-A) sequence, inducing TdT-mediated polymerization and the subsequent hybridization with more poly-A signal probes for generating dendritic DNA nanostructures assembled on MBs. After magnetic separation and elevated temperature treatment, the signal probes are disassembled from MBs to generate a high fluorescence signal. This method possesses excellent specificity and high sensitivity with a limit of detection (LOD) of 2.61 × 10-15 M, and it can accurately quantify cellular METTL3-METTL14 complex at single-cell level. Furthermore, it can screen inhibitors, evaluate kinetic parameters, and discriminate breast cancer tissues from normal tissues.Copyright © 2023 Elsevier B.V. All rights reserved.