He Jing-Lin, Mei Ting-Ting, Tang Ling, Liao Shi-Qing, Cao Zhong
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, 410114, PR China.
Hunan Provincial Key Laboratory of Materials Protection for Electric Power and Transportation, Hunan Provincial Key Laboratory of Cytochemistry, School of Chemistry and Food Engineering, Changsha University of Science and Technology, Changsha, 410114, PR China.
Talanta. 2020 Nov 1;219:121173. doi: 10.1016/j.talanta.2020.121173. Epub 2020 May 23.
Sensitive and specific detection of microRNAs (miRNAs) is of great significance for early cancer diagnosis. Here we report a simple and sensitive fluorescence signal amplification strategy that based on DSN/TdT recycling digestion for miRNA detection. DSN initiates DNA digestion on 3'-phosphate-primer/miRNA heteroduplex which causes miRNA recycle. The digested DNA strands with 3'-OH ends enable TdT to synthesize a polydeoxyguanylic tails on the 3'-end. The DNAs with polydeoxyguanylic tails are converted to double-stranded-DNA prior to initiation of DSN/TdT recycling digestion. With the cooperation of TdT and DSN, a new round of digestion and extension is triggered, leading to massive fluorophores separating and signal amplification. The amplification strategy produces large amounts of 3'-OH probes that can be used directly for dsDNA enrichment and DSN digestion. Moreover, both DSN digestion and TdT extension are sequence-independent reaction without the need of complex sequences design. In addition, this strategy is utilized to analyze miRNA samples from MCF-7 cell lysates and Cu (II) ion samples, indicating its potential application in actual sample analysis. The method shows a promising analytical platform for DNA nicking-related studies and tumor biomarkers measuring in clinical diagnostics.
对微小RNA（miRNA）进行灵敏且特异的检测对于癌症早期诊断具有重要意义。在此，我们报告一种基于DSN/TdT循环消化用于miRNA检测的简单且灵敏的荧光信号放大策略。DSN启动对3'-磷酸引物/miRNA异源双链体的DNA消化，从而导致miRNA循环。具有3'-OH末端的消化后的DNA链使TdT能够在3'-末端合成聚脱氧鸟苷酸尾巴。在启动DSN/TdT循环消化之前，将具有聚脱氧鸟苷酸尾巴的DNA转化为双链DNA。在TdT和DSN的协同作用下，引发新一轮的消化和延伸，导致大量荧光团分离并实现信号放大。该放大策略产生大量可直接用于双链DNA富集和DSN消化的3'-OH探针。此外，DSN消化和TdT延伸均为不依赖序列的反应，无需复杂的序列设计。此外，该策略被用于分析MCF-7细胞裂解物中的miRNA样品和铜（II）离子样品，表明其在实际样品分析中的潜在应用。该方法为DNA切口相关研究和临床诊断中的肿瘤生物标志物检测提供了一个有前景的分析平台。
