A one-pot isothermal Cas12-based assay for the sensitive detection of microRNAs.

Abstract

The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, fast and inexpensive assays for their detection in biofluids. Here we report a one-step and one-pot isothermal assay that leverages rolling-circle amplification and the endonuclease Cas12a for the accurate detection of specific miRNAs. The assay exploits the cis-cleavage activity of Cas12a to enable exponential rolling-circle amplification of target sequences and its trans-cleavage activity for their detection and for signal amplification. In plasma from patients with pancreatic ductal adenocarcinoma, the assay detected the miRNAs miR-21, miR-196a, miR-451a and miR-1246 in extracellular vesicles at single-digit femtomolar concentrations with single-nucleotide specificity. The assay is rapid (sample-to-answer times ranged from 20 min to 3 h), does not require specialized instrumentation and is compatible with a smartphone-based fluorescence detection and with the lateral-flow format for visual readouts. Simple assays for the detection of miRNAs in blood may aid the development of miRNAs as biomarkers for the diagnosis and prognosis of cancers.

• Authors: He Yan, Yunjie Wen, Zimu Tian, Nathan Hart, Song Han, Steven J Hughes, Yong Zeng
• Journal: Nature biomedical engineering (Nat Biomed Eng) Vol. 7 Issue 12 Pg. 1583-1601 (Dec 2023) ISSN: 2157-846X [Electronic] England
• PMID: 37106152 (Publication Type: Journal Article)
• Copyright: © 2023. The Author(s), under exclusive licence to Springer Nature Limited.

Chemical References

• MicroRNAs
• Biomarkers, Tumor

Topics

• Humans
• MicroRNAs
• Biomarkers, Tumor (genetics)
• Pancreatic Neoplasms (diagnosis, genetics)
• Prognosis