Key Laboratory of Optoelectronic Chemical Materials and Devices of Ministry of Education, College of Photoelectric Materials and Technology, Jianghan University, Wuhan 430056, PR China.
Department of Respiratory and Critical Care Medicine, Key Laboratory of Pulmonary Diseases of Health Ministry, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430056, PR China.
Anal Methods. 2021 Dec 9;13(47):5694-5699. doi: 10.1039/d1ay01709h.
MicroRNAs play important roles in disease diagnosis and therapy. However, current methods for microRNA detection suffer from low sensitivity and cannot directly detect short microRNAs. Herein, we have developed a highly sensitive and selective fluorescent method for direct microRNA detection by combining the duplex-specific nuclease-assisted recycling amplification and the nicking enzyme-powered three-dimensional DNA walker. Target microRNA initiates duplex-specific nuclease-assisted recycling amplification, releasing numerous bipedal walking strands. The released bipedal walking strands hybridize with carboxyfluorescein-labeled track DNA and form nicking recognition site. Driven by the hydrolysis of the nicking enzyme, the bipedal walking strand autonomously moves along the track strand, releasing a large number of carboxyfluorescein-labeled DNA fragments and generating obvious fluorescence signals. This dual-signal amplification method can directly detect microRNA 21 as low as 130 fM and has good selectivity. The proposed method is not only simple for nucleic acid design, but also can be used as a universal method for the highly sensitive detection of all RNAs.
MicroRNAs 在疾病诊断和治疗中发挥着重要作用。然而，目前的 microRNA 检测方法灵敏度低，无法直接检测短 microRNAs。在此，我们结合双链特异性核酸酶辅助的循环放大和缺口酶驱动的三维 DNA 行走，开发了一种用于直接 microRNA 检测的高灵敏度和选择性荧光方法。靶 microRNA 启动双链特异性核酸酶辅助的循环放大，释放出大量双足行走链。释放的双足行走链与羧基荧光素标记的轨道 DNA 杂交，并形成缺口识别位点。在切口酶的水解作用下，双足行走链沿着轨道链自动移动，释放大量羧基荧光素标记的 DNA 片段并产生明显的荧光信号。这种双信号扩增方法可以直接检测低至 130 fM 的 microRNA 21，具有良好的选择性。所提出的方法不仅在核酸设计上简单，而且可以作为一种通用的方法，用于所有 RNA 的高灵敏度检测。
