Zhao Ning-Ning, Zhang Xinyi, Zou Xiaoran, Zhang Yan, Zhang Chun-Yang
College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
School of Chemistry and Chemical Engineering, Guangdong Pharmaceutical University, Zhongshan, 528458, China.
Biosens Bioelectron. 2023 May 15;228:115217. doi: 10.1016/j.bios.2023.115217. Epub 2023 Mar 11.
N-Methyladenosine (mA) 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 mA 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 mA 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 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.
N - 甲基腺苷（mA）是真核生物信使核糖核酸（mRNA）和长链非编码核糖核酸（lncRNA）中的一种可逆化学修饰。RNA甲基转移酶METTL3 - METTL14复合物的异常表达可能会改变mA甲基化水平，并引发包括癌症在内的多种疾病。传统的METTL3 - METTL14检测方法通常耗时且灵敏度低。在此，我们基于MazF激活的末端脱氧核苷酸转移酶（TdT）辅助的树枝状DNA结构组装开发了一种可控扩增机制，用于超灵敏检测癌细胞和乳腺组织中的METTL3 - METTL14复合物活性。METTL3 - METTL14复合物的存在催化检测探针中mA的形成，有效防止MazF对甲基化检测探针的切割。具有3' - OH末端的甲基化检测探针可作为TdT在磁珠（MBs）上催化的无模板聚合反应的引物，产生长链的聚胸腺嘧啶（poly - T）序列。然后，poly - T序列与含有聚腺嘌呤（poly - A）序列的信号探针杂交，诱导TdT介导的聚合反应以及随后与更多poly - A信号探针的杂交，从而在MBs上生成组装的树枝状DNA纳米结构。经过磁分离和高温处理后，信号探针从MBs上解离，产生高荧光信号。该方法具有出色的特异性和高灵敏度，检测限（LOD）为2.61×10⁻⁹ M，并且能够在单细胞水平上准确量化细胞中的METTL3 - METTL14复合物。此外，它还可以筛选抑制剂、评估动力学参数，并区分乳腺癌组织和正常组织。
