Kim Minhee, Kim Dong-Min, Kim Dong-Eun
Department of Bioscience and Biotechnology, Konkuk University, Gwangjin-gu, Seoul 05029, Republic of Korea.
Analyst. 2020 Sep 14;145(18):6130-6137. doi: 10.1039/d0an01329c.
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.
微小RNA（miRNA）在各种生物学过程中发挥着重要作用，在临床诊断领域已被视为实体瘤有前景的诊断生物标志物。在本研究中，我们开发了一种简单的无标记/无淬灭荧光系统，用于使用等温基因扩增（如滚环扩增产生串联G-四链体DNA结构，即GQ-RCA）灵敏且选择性地检测miRNA。封闭环状哑铃形锁式DNA被设计成与其相应的靶miRNA互补。在靶miRNA存在的情况下，phi29 DNA聚合酶通过在与锁式DNA退火的靶miRNA的3'-OH处引发DNA合成，由RCA容易地产生具有串联G-四链体序列重复的长链单链DNA。携带串联G-四链体的RCA产物用荧光团硫黄素T（ThT）进行监测，ThT仅在插入G-四链体时才发出强荧光。GQ-RCA分析使我们能够在0.5小时内检测低至4.9 fM的miRNA，线性范围为25.6 fM至80 pM。此外，我们的系统应用于人类血浆中存在的miRNA样本，显示出其在癌症临床诊断中的潜在用途。
