A Cas12a-mediated cascade amplification method for microRNA detection

MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans -cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detec...

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Bibliographic Details
Published in:Analyst (London) 2020-08, Vol.145 (16), p.5547-5552
Main Authors: Sun, Huan-Huan, He, Fang, Wang, Ting, Yin, Bin-Cheng, Ye, Bang-Ce
Format: Article
Language:English
Subjects:
Amplification
Biological activity
Biomarkers
CRISPR-Cas Systems
DNA
DNA, Catalytic
Fluorescence
Homology
MicroRNAs
MicroRNAs - genetics
Nucleic Acid Amplification Techniques
Ribonucleic acid
RNA
Strands
Target detection
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Summary:MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy. Here, we took advantage of Cas12a trans -cleavage activity to develop an enzyme-assisted cascade amplification method for isothermal miRNA detection. A target miRNA-initiated ligation reaction would allow for the production of transcription templates that triggered the transcriptional amplification of RNA strands. These RNA strands were cleaved by the 8-17E DNAzyme to generate crRNAs and recycled RNAs which have the same sequence as the target miRNA. The amplified abundant crRNAs bound to Cas12a and dsDNA activators to form the complex, which trans -cleaved the ssDNA reporters to generate a fluorescence signal for miRNA quantitative analysis. The proposed method exhibits a femtomolar limit of detection and a good specificity in distinguishing the homologous sequences of miRNAs. Its practical application ability was further tested in different cell lines. MicroRNAs (miRNAs) play a vital role in various biological processes and act as important biomarkers for clinical cancer diagnosis, prognosis, and therapy.
ISSN:0003-2654
1364-5528
DOI:10.1039/d0an00370k