Liu Qian, Zhou Bao-Mei, Wang Li-Juan, Zhang Chun-Yang
School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing, 211189, China.
School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing, 211189, China.
Biosens Bioelectron. 2025 Apr 15;274:117198. doi: 10.1016/j.bios.2025.117198. Epub 2025 Jan 28.
N-methyladenosine (mA) is the most important internal methylation in eukaryotic RNAs, and it is critically implicated in diverse RNA metabolisms for cancer development. Because epigenetic modifications do not interfere with Watson-Crick base pairing and mA modification is not susceptible to chemical decorations, standard hybridization-based techniques cannot be applied for sensing mA in RNAs. Consequently, the development of new methods for accurate and sensitive profiling of locus-specific mA in RNAs remains a great challenge. Herein, we demonstrate for the first time the construction of a hierarchical DNA circuit for single-molecule profiling of locus-specific mA-metastasis-associated lung adenocarcinoma transcript 1 (mA-MALAT1) in clinical tissues. Taking advantage of high discrimination of VMC10-DNAzyme between mA and A, exponential efficiency of hierarchical DNA circuit, and ultrahigh signal-to-noise ratio of single-molecule detection, this nanodevice exhibits attomolar sensitivity with a limit of detection (LOD) of 1.8 aM for mA-MALAT1 in vitro and a dynamic range of 7 orders of magnitude. Moreover, it can discriminate 0.001% mA-MALAT1 from excess A-MALAT1, quantify mA-MALAT1 in diverse cancer cells at single-cell level, distinguish mA-MALAT1 expressions in breast cancer patients and healthy individuals, and monitor cellular mA-MALAT1 for gene therapy, offering a promising platform for epitranscriptomic research and clinical diagnostics.
N6-甲基腺苷（m6A）是真核生物RNA中最重要的内部甲基化修饰，在癌症发生发展的多种RNA代谢过程中起关键作用。由于表观遗传修饰不干扰沃森-克里克碱基配对，且m6A修饰不易受化学修饰影响，基于标准杂交的技术无法用于检测RNA中的m6A。因此，开发准确、灵敏地分析RNA中位点特异性m6A的新方法仍然是一项巨大挑战。在此，我们首次展示了构建一种分级DNA电路，用于对临床组织中位点特异性m6A-转移相关肺腺癌转录本1（m6A-MALAT1）进行单分子分析。利用VMC10-DNAzyme对m6A和腺嘌呤（A）的高区分能力、分级DNA电路的指数放大效率以及单分子检测的超高信噪比，该纳米装置在体外对m6A-MALAT1表现出飞摩尔灵敏度，检测限（LOD）为1.8 aM，动态范围达7个数量级。此外，它能够从过量的A-MALAT1中区分出0.001%的m6A-MALAT1，在单细胞水平定量多种癌细胞中的m6A-MALAT1，区分乳腺癌患者和健康个体中m6A-MALAT1的表达情况，并监测细胞内m6A-MALAT1用于基因治疗，为表观转录组学研究和临床诊断提供了一个有前景的平台。
