Department of Radiology, Xinqiao Hospital, Army Medical University, Chongqing, 400038, China.
Department of Radiology, Chongqing Kanghua Zhonglian Cardiovascular Hospital, Chongqing, 400025, China.
Anal Biochem. 2022 Oct 1;654:114744. doi: 10.1016/j.ab.2022.114744. Epub 2022 May 25.
MicroRNA (miRNA) and circular RNA (circRNA) are promising biomarkers for early-diagnosis of a variety of diseases, such as myocardial infarction and cancers. However, few methods reported simultaneous and sensitive detection of multiple miRNAs. Herein, we design a novel approach with an improved miRNA and circRNA detection sensitivity. There are only two probes designed with hairpin probe, namely catch probe and report probe, in the system. When target miRNA or circRNA existed, it can unfold catch probe through hybridizing with toehold section and form a rigid DNA-RNA duplex. The DNA sequence in the formed duplex is identified and digested by duplex-specific nuclease (DSN enzyme), and consequently, target miRNA is released to attend a next signal cycle. The rested DNA sequence in catch probe (initiator probe) recognizes report probe and separates its hairpin structure to generate fluorescence signals, forming a double-strand DNA (dsDNA) product. Unfold report probe sequence in the dsDNA product is then digested by DNA nicking endonuclease (NEase) and initiator is released to trigger amplified signals. Based on the DSN enzyme cooperating NEase assisted dual signal amplification, the method exhibits a greatly improved detection sensitivity.
MicroRNA (miRNA) 和 circular RNA (circRNA) 是多种疾病（如心肌梗死和癌症）早期诊断有前途的生物标志物。然而，很少有方法能够同时灵敏地检测多种 miRNA。在此，我们设计了一种新方法，可提高 miRNA 和 circRNA 的检测灵敏度。该系统中仅设计了两个带有发夹探针的探针，即捕获探针和报告探针。当存在靶 miRNA 或 circRNA 时，它可以通过与结合点杂交展开捕获探针，并形成刚性 DNA-RNA 双链。形成的双链中的 DNA 序列被双链特异性核酸酶（DSN 酶）识别并消化，从而释放靶 miRNA 以参与下一个信号循环。捕获探针（启动子探针）中的休息 DNA 序列识别报告探针并分离其发夹结构以产生荧光信号，形成双链 DNA (dsDNA) 产物。dsDNA 产物中报告探针序列的展开随后被 DNA 切口内切酶 (NEase) 消化，并释放启动子以触发扩增信号。基于 DSN 酶协同 NEase 辅助的双信号扩增，该方法表现出大大提高的检测灵敏度。
