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The trans-cleavage activity of the Cas12a protein is triggered by the formation of complete hairpin-shaped CRT products; this results in the cleavage of the DNA section contained in the H probe, while the RNA section (“4”@MBs) remains loaded onto the surface of magnetic beads (MB). By binding with the “reporter” sensor, the “4” sequences create an RNA/DNA duplex that the duplex-specific nuclease (DSN) can recognize. The “reporter” probe is thus metabolized, leading to the reappearance of the fluorescence signal. By capitalizing on the exceptional fidelity and selectivity of CRISPR/Cas12a, as well as the substantial impact of triggered enzymatic cycle amplification, this approach demonstrated remarkable sensitivity and specificity in miRNA detection, even in a complex environment containing 10% fetal bovine serum (FBS) and a serum sample. In contrast, a detection limit of 3.2 fM is conceivable. 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引用次数: 0
摘要
微小核糖核酸(miRNA)是一类具有重要调控能力的小分子,已被广泛用作生物传感领域的生物标志物,以促进对各种疾病的早期检测。然而,由于同源序列之间的高度相似性和低丰度,灵敏而准确的 miRNA 检测仍然是一个巨大的挑战。因此,开发具有高灵敏度和特异性的 miRNA 检测方法至关重要。在本研究中,我们利用循环反转录(CRT)和CRISPR-Cas12a开发了一种基于信号循环的平台,实现了对microRNA的精确、灵敏检测。CRT 机制有助于在目标 miRNA 存在的情况下精确识别目标,从而将 miRNA 信号转换为 DNA 信号。Cas12a 蛋白的反式裂解活性是通过形成完整的发夹形 CRT 产物触发的;这会导致 H 探针所含的 DNA 部分被裂解,而 RNA 部分("4"@MBs)仍被载入磁珠(MB)表面。通过与 "报告 "传感器结合,"4 "序列产生了一个 RNA/DNA 双链,双链特异性核酸酶(DSN)可以识别该双链。这样,"报告 "探针就会被代谢掉,从而重新出现荧光信号。通过利用 CRISPR/Cas12a 的优异保真度和选择性,以及触发酶循环扩增的重大影响,这种方法在 miRNA 检测方面表现出了显著的灵敏度和特异性,即使在含有 10% 胎牛血清(FBS)和血清样本的复杂环境中也是如此。相比之下,3.2 fM 的检测限是可以想象的。此外,这种方法还保持了显著的稳定性,预计能以有效、灵敏的方式检测 miRNA。
Sensitive and reliable miRNA analysis based on cyclic reverse transcription and CRISPR-Cas12a-assisted signal cycle
MicroRNAs (miRNAs), a category of small molecules that possess significant regulatory capabilities, have been extensively employed as biomarkers in the domain of biosensing to facilitate the early detection of diverse ailments. However, sensitive and accurate miRNA detection remains a huge challenge due to the high similarity between the homologous sequences and low abundance. Therefore, it is essential to develop methods with high sensitivity and specificity for miRNA detection. In this study, we present the development of a signal cycle-based platform that utilizes cyclic reverse transcription (CRT) and CRISPR-Cas12a to enable the precise and sensitive detection of microRNAs. The CRT mechanism facilitates precise target recognition in the presence of target miRNA, thereby converting miRNA signals to DNA signals. The trans-cleavage activity of the Cas12a protein is triggered by the formation of complete hairpin-shaped CRT products; this results in the cleavage of the DNA section contained in the H probe, while the RNA section (“4”@MBs) remains loaded onto the surface of magnetic beads (MB). By binding with the “reporter” sensor, the “4” sequences create an RNA/DNA duplex that the duplex-specific nuclease (DSN) can recognize. The “reporter” probe is thus metabolized, leading to the reappearance of the fluorescence signal. By capitalizing on the exceptional fidelity and selectivity of CRISPR/Cas12a, as well as the substantial impact of triggered enzymatic cycle amplification, this approach demonstrated remarkable sensitivity and specificity in miRNA detection, even in a complex environment containing 10% fetal bovine serum (FBS) and a serum sample. In contrast, a detection limit of 3.2 fM is conceivable. Furthermore, this approach maintained a notable degree of stability, which was anticipated to result in the detection of miRNAs in an effective and sensitive manner.
期刊介绍:
The Journal of Analytical Science and Technology (JAST) is a fully open access peer-reviewed scientific journal published under the brand SpringerOpen. JAST was launched by Korea Basic Science Institute in 2010. JAST publishes original research and review articles on all aspects of analytical principles, techniques, methods, procedures, and equipment. JAST’s vision is to be an internationally influential and widely read analytical science journal. Our mission is to inform and stimulate researchers to make significant professional achievements in science. We aim to provide scientists, researchers, and students worldwide with unlimited access to the latest advances of the analytical sciences.