Ultrasensitive detection of microRNAs based on click chemistry- terminal deoxynucleotidyl transferase combined with CRISPR/Cas12a

文献类型： 外文期刊

第一作者： Li, Xue

作者： Li, Xue;Liu, Xiaoyu;Wei, Jiaqi;Zhang, Wenhui;Li, Xue;Liu, Xiaoyu;Wei, Jiaqi;Bu, Shengjun;Li, Zhongyi;Hao, Zhuo;Wan, Jiayu

作者机构：

关键词： microRNA; TdT; Click chemistry; CRISPR; Cas12a

期刊名称：BIOCHIMIE （ 影响因子：3.9； 五年影响因子：3.9 ）

ISSN： 0300-9084

年卷期： 2023 年 208 卷

页码：

收录情况： SCI

摘要： The specificity and sensitivity of microRNA (miRNA) detection play a vital role in the early diagnosis of cancer and the treatment of various diseases. Here, we constructed a fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) combined with the clustered regularly inter-spaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)12a cascade amplification system to achieve ultrasensitive miRNA-21 detection. Target miRNA-21 was employed as a template for click chemistry ligation of two nucleic acid probes, the product of which can be combined with magnetic microbeads (MBs). Then the 30-end of the ligated nucleic acid and complementary strand miRNA-21 was extended by TdT. The extended poly-T tails activated the trans-cleavage ability of CRISPR/Cas12a, cleaving the reporter gene to generate the fluorescent signal. The proposed biosensor has a wide linear detection range, from 1 pM to 105 pM, with detection limits as low as 88 fM under optimal experimental condi-tions. Hence, this fluorescent biosensor enables simple, sensitive detection of miRNAs and offers a promising analytical platform for clinical diagnostics and biomedical research.(c) 2022 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

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