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具有更高准确性的双靶点识别介导的核酸外切酶III(Exo-III)驱动的级联信号循环微小RNA检测系统。

Twice target-recognition mediated exonuclease iii (Exo-iii)-Propelled cascade signal recycling MicroRNA detection system with improved accuracy.

作者信息

Jia Yuling, Yuan Jianhua, Zheng Yanlei, Huang Yanzhen, Zhang Juncai, Zhao Haibin, Zhang Jiefang

机构信息

Cardiovascular Medicine Department, Shijiazhuang Fourth Hospital (Obstetrics and Gynecology Hospital Affiliated to Hebei Medical University), Shijiazhuang City, Hebei Province, 050033, China.

Cardiovascular Medicine Department, Gucheng County Hospital, Hengshui City, Hebei Province, 253800, China.

出版信息

Anal Biochem. 2025 Apr;699:115757. doi: 10.1016/j.ab.2024.115757. Epub 2024 Dec 30.

Abstract

Simple yet specific miRNA detection remains an enormous challenge due to its low abundance in samples and the high similarity among homologous miRNAs. Here, we propose a novel fluorescent approach for miRNA detection with greatly elevated accuracy by utilizing exonuclease-iii (Exo-iii) assisted twice target recognition. The proposed method involves a "Sensing probe" engineered with two loop sections to facilitate dual target miRNA recognition. The collaboration between Exo-iii and miRNA initiates target recycling for signal amplification, resulting in the formation of complete DNAzyme. The intact DNAzyme connects with the "Signal probe" and creates a nicking site within its loop region. The fluorescence signal of the "Signal probe" reemerges, correlating with the quantity of miRNA in the sensing system. The suggested technique demonstrates great selectivity for target miRNA and can readily differentiate sequences with a one-base mismatch, based on dual-target identification. Furthermore, the Exo-iii-assisted signal recycling imparts the approach with great sensitivity and a low detection limit of 548 aM. This method has the potential to be a robust alternative for the detection of miRNAs in real samples due to its high accuracy, simplicity, and resistance to potential fluorescence interferences.

摘要

由于样本中微小RNA(miRNA)丰度低且同源miRNA之间相似度高,简单而特异的miRNA检测仍然是一项巨大挑战。在此,我们提出一种新型荧光方法用于miRNA检测,通过利用核酸外切酶III(Exo-III)辅助的两次靶标识别,显著提高了检测准确性。所提出的方法涉及一种“传感探针”,其设计有两个环结构域以促进对双重靶标miRNA的识别。Exo-III与miRNA之间的协同作用启动靶标循环以进行信号放大,从而形成完整的脱氧核酶。完整的脱氧核酶与“信号探针”连接,并在其环区域内产生一个切口位点。“信号探针”的荧光信号重新出现,与传感系统中miRNA的数量相关。所建议的技术对靶标miRNA具有高度选择性,基于双重靶标识别,能够轻松区分单碱基错配的序列。此外,Exo-III辅助的信号循环赋予该方法高灵敏度和548 aM的低检测限。由于其高准确性、简单性以及对潜在荧光干扰的抗性,该方法有潜力成为检测实际样本中miRNA的强大替代方法。

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