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

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.

Automated summary

A fluorescent biosensor based on click chemistry-terminal deoxynucleotidyl transferase (ccTdT) and the CRISPR/Cas12a cascade amplification system was constructed for ultrasensitive miRNA-21 detection, providing a promising platform for clinical diagnostics and biomedical research.