A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing
| Autor: | Xiufeng Gan, Decai Zhang, Tiantian Yang, Qingyuan Zheng, Xingrong Li, Ping Liu, Yurong Yan, Guozhen Tian, Shijia Ding |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
CRISPR-Associated Proteins Biomedical Engineering Blood Donors Biosensing Techniques Computational biology Cleavage (embryo) Sensitivity and Specificity 01 natural sciences Biochemistry Genetics and Molecular Biology (miscellaneous) Fluorescence 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins 010608 biotechnology microRNA Humans CRISPR Clustered Regularly Interspaced Short Palindromic Repeats 030304 developmental biology Gene Editing 0303 health sciences Endodeoxyribonucleases Nucleic Acid Hybridization DNA General Medicine Reverse transcriptase Nanostructures Highly sensitive MicroRNAs Linear range chemistry Cascade MCF-7 Cells CRISPR-Cas Systems Nucleic Acid Amplification Techniques HeLa Cells |
| Zdroj: | ACS Synthetic Biology. 10:1481-1489 |
| ISSN: | 2161-5063 |
| DOI: | 10.1021/acssynbio.1c00064 |
| Popis: | The variations of microRNA (miRNA) expression can be valuable biomarkers in disease diagnosis and prognosis. However, current miRNA detection techniques mainly rely on reverse transcription and template replication, which suffer from slowness, contamination risk, and sample loss. To address these limitations, here we introduce a cascade toehold-mediated strand displacement reaction (CTSDR) and CRISPR/Cas12a trans-cleavage for highly sensitive fluorescent miRNA sensing, namely CTSDR-Cas12a. In this work, the target miRNA hybridizes with the terminal toehold site of a rationally designed probe and subsequently initiates dynamic CTSDR, leading to enzyme-free target recycling and the production of multiple programmable DNA duplexes. The obtained DNA duplex acts as an activator to trigger Cas12a trans-cleavage, generating significantly amplified fluorescence readout for highly sensitive detection of the miRNA target. Under the optimal conditions, the developed sensing method can detect target miRNA down to 70.28 fM with a wide linear range from 100 fM to 100 pM. In particular, by designing a set of probes and crRNAs, we demonstrate its broad applicability for the detection of six kinds of miRNAs with high sequence specificity. Furthermore, the method can be satisfactorily applied to monitor miR-21 in total RNA extracted from cells and clinical serum samples. Considering the high sensitivity, specificity, universality, and ease of handling, this strategy provides a great potential platform for the detection of miRNA biomarkers in molecular diagnostic practice. |
| Databáze: | OpenAIRE |
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