"DSN-mismatched CRISPR″sensor for highly selective and sensitive detection of under-expressed miR-let-7a.

Autor: Qin H; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China; Department of Clinical Laboratory, Dazhou Central Hospital, Dazhou, 635000, PR China., Chen Z; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China., Zuo F; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China., Cao R; College of Basic Medicine, Zunyi Medical University, Zunyi, 563000, PR China., Wang F; College of Basic Medicine, Zunyi Medical University, Zunyi, 563000, PR China., Wu H; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China., Wang S; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China., Xie Y; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China., Ding S; Key Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, PR China., Min X; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China. Electronic address: zmchj2001@163.com., Duan X; Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, 563000, PR China; College of Basic Medicine, Zunyi Medical University, Zunyi, 563000, PR China. Electronic address: duanxiaolei0@163.com.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2024 Mar 22; Vol. 1295, pp. 342273. Date of Electronic Publication: 2024 Jan 27.
DOI: 10.1016/j.aca.2024.342273
Abstrakt: Several microRNAs (miRNAs) are expressed at lower levels in specific tumors, e.g., miR-let-7a in non-small cell lung cancer (NSCLC). This makes it challenging to analyze their lower abundance versus specifically elevated miRNAs. Here, we describe a novel fluorescent biosensor for the highly selective and sensitive detection of miR-let-7a constructed by combining miRNA screening assisted by a duplex-specific nuclease (DSN) with CRISPR-Cas12a system signal amplification. We meticulously designed a mismatch in the first three to four bases at the 5'-end of the capture DNA to improve the signal-to-noise ratio of the CRISPR-Cas12a system. Within this "DSN-mismatched CRISPR" fluorescence strategy, miR-let-7a was accurately screened by DSN-assisted cleavage, and the mismatched capture DNA unbound to target miRNA could trigger the CRISPR-Cas12a system to produce a mass of trans-cleave fluorescence signals. This "turn-off" approach was suitable for detecting decreased levels of miRNAs. This approach can not only discriminate the single-base mismatched let-7 family but also reach a limit of detection at 64.17 fM as well as be quantified from 100 fM to 500 pM. The miR-let-7a levels were then measured in clinical serum samples from healthy volunteers and patients with NSCLC. This study holds promise for the development of a universal under-expressed miRNA assay for early diagnosis and treatment of cancers.
Competing Interests: Declaration of competing interest This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal. We have read and understood your journal's policies, and we believe that neither the manuscript nor the study violates any of these. There are no conflicts of interest to declare.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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