Sun Huan-Huan, He Fang, Wang Ting, Yin Bin-Cheng, Ye Bang-Ce
Laboratory of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Analyst. 2020 Aug 21;145(16):5547-5552. doi: 10.1039/d0an00370k. Epub 2020 Jul 1.
MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans-cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detection. A target miRNA-initiated ligation reaction would allow for the production of transcription templates that triggered the transcriptional amplification of RNA strands. These RNA strands were cleaved by the 8-17E DNAzyme to generate crRNAs and recycled RNAs which have the same sequence as the target miRNA. The amplified abundant crRNAs bound to Cas12a and dsDNA activators to form the complex, which trans-cleaved the ssDNA reporters to generate a fluorescence signal for miRNA quantitative analysis. The proposed method exhibits a femtomolar limit of detection and a good specificity in distinguishing the homologous sequences of miRNAs. Its practical application ability was further tested in different cell lines.
微小RNA（miRNA）在各种生物学过程中发挥着至关重要的作用，并作为临床癌症诊断、预后和治疗的重要生物标志物。在此，我们利用Cas12a的反式切割活性开发了一种用于等温miRNA检测的酶辅助级联扩增方法。靶miRNA引发的连接反应将允许产生触发RNA链转录扩增的转录模板。这些RNA链被8-17E DNAzyme切割以产生与靶miRNA具有相同序列的crRNA和循环RNA。扩增产生的大量crRNA与Cas12a和dsDNA激活剂结合形成复合物，该复合物反式切割单链DNA报告分子以产生用于miRNA定量分析的荧光信号。所提出的方法具有飞摩尔检测限，并且在区分miRNA的同源序列方面具有良好的特异性。其实际应用能力在不同细胞系中进一步得到测试。
