A Cascade Signal Amplification Based on Dynamic DNA Nanodevices and CRISPR/Cas12a Trans-cleavage for Highly Sensitive MicroRNA Sensing

作者全名："Li, Xingrong; Zhang, Decai; Gan, Xiufeng; Liu, Ping; Zheng, Qingyuan; Yang, Tiantian; Tian, Guozhen; Ding, Shijia; Yan, Yurong"

作者地址："[Li, Xingrong; Gan, Xiufeng; Liu, Ping; Zheng, Qingyuan; Ding, Shijia; Yan, Yurong] Chongqing Med Univ, Coll Lab Med, Minist Educ, Key Lab,Clin Lab Diagnost, Chongqing 400016, Peoples R China; [Zhang, Decai; Yang, Tiantian] Chongqing Med Univ, Ctr Clin Mol Med Detect, Affiliated Hosp 1, Chongqing 400016, Peoples R China; [Zhang, Decai] Shenzhen Univ, Affiliated Hosp 3, Dept Lab Diag, Shenzhen 518000, Peoples R China; [Tian, Guozhen] Hainan Med Univ, Sch Trop Med & Lab Med, Key Lab Trop Translat Med, Minist Educ, Haikou 571199, Hainan, Peoples R China"

通信作者："Yan, YR (corresponding author), Chongqing Med Univ, Coll Lab Med, Minist Educ, Key Lab,Clin Lab Diagnost, Chongqing 400016, Peoples R China."

来源：ACS SYNTHETIC BIOLOGY

ESI学科分类：BIOLOGY & BIOCHEMISTRY

WOS号：WOS:000664354700022

JCR分区：Q1

影响因子：4.7

年份：2021

卷号：10

期号：6

开始页：1481

结束页：1489

文献类型：Article

关键词：CRISPR/Cas12a; PAM sequence; dynamic DNA nanodevices; cascade toehold-mediated strand displacement reaction; MicroRNA detection

摘要："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."

基金机构："National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [81371904, 81873980]; National Science and Technology Major Project of the Ministry of Science and Technology of China [2018ZX10732202]; Natural Science Foundation Project of ChongqingNatural Science Foundation of Chongqing [cstc2018jcyjAX0349]"

基金资助正文："This work was funded by financial support from the National Natural Science Foundation of China (81371904, 81873980), the National Science and Technology Major Project of the Ministry of Science and Technology of China (2018ZX10732202), and the Natural Science Foundation Project of Chongqing (cstc2018jcyjAX0349)."