MicroRNAs (miRNAs) play an important role in various biological processes and have been regarded as promising diagnostic biomarkers for solid tumors in the field of clinical diagnostics. In this study, we developed a simple label/quencher-free fluorometric system for sensitive and selective miRNA detection using isothermal gene amplification such as rolling circle amplification generating tandem G-quadruplex DNA structures (GQ-RCA). The closed-circular dumbbell-shaped padlock DNA was designed to be complementary to its corresponding target miRNA. In the presence of the target miRNA, a long stretch of ssDNA with tandem G-quadruplex sequence repeats was readily generated by RCA, initiated by phi29 DNA polymerase through DNA synthesis priming at the 3′-OH of the target miRNA annealed to the padlock DNA. The RCA product harboring tandem G-quadruplex was monitored with fluorophore Thioflavin T (ThT) that emits strong fluorescence only when it intercalates into the G-quadruplex. The GQ-RCA assay enabled us to detect miRNA as low as 4.9 fM with a linear range from 25.6 fM to 80 pM within 0.5 h. In addition, our system was applied to the miRNA samples present in human plasma, showing its potential use in the clinical diagnosis of cancer.

中文翻译：

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使用等温滚动环扩增产生串联G-四链体，对microRNA进行无标记的荧光检测。

微小RNA（miRNA）在各种生物学过程中起着重要作用，在临床诊断领域已被视为有前途的实体瘤诊断生物标志物。在这项研究中，我们开发了一种简单的无标记/无猝灭剂的荧光测定系统，用于使用等温基因扩增（如产生串联G-四链体DNA结构（GQ-RCA）的滚环扩增）进行灵敏和选择性的miRNA检测。闭合圆形哑铃状的挂锁DNA被设计为与其相应的靶标miRNA互补。在目标miRNA存在的情况下，RCA容易生成带有串联G-四链体序列重复序列的长片段ssDNA，这是由phi29 DNA聚合酶通过DNA合成引发的，在退火至挂锁DNA的目标miRNA的3'-OH上引发的。 。用荧光团硫黄素T（ThT）监测带有串联G-四链体的RCA产物，该荧光团只有插入到G-四链体中时才会发出强荧光。GQ-RCA分析使我们能够在0.5 h内检测到范围从25.6 fM到80 pM的低至4.9 fM的miRNA。此外，我们的系统已应用于人血浆中存在的miRNA样品，显示了其在癌症临床诊断中的潜在用途。