A novel fluorescence amplification strategy combining cascade primer exchange reaction with CRISPR/Cas12a system for ultrasensitive detection of RNase H activity

作者全名："Xie, Zuowei; Chen, Siyi; Zhang, Wenxiu; Zhao, Shuhui; Zhao, Zixin; Wang, Xingyu; Huang, Yuqi; Yi, Gang"

作者地址："[Xie, Zuowei; Chen, Siyi; Zhang, Wenxiu; Zhao, Shuhui; Zhao, Zixin; Yi, Gang] Chongqing Med Univ, Coll Lab Med, Key Lab Med Diagnost, Minist Educ, Chongqing 400016, Peoples R China; [Wang, Xingyu] Wuxi Peoples Hosp, Clin Lab, Chongqing 405800, Peoples R China; [Huang, Yuqi] Jiulongpo Peoples Hosp, Clin Lab, Chongqing 400050, PR, Peoples R China"

通信作者："Yi, G (通讯作者)，Chongqing Med Univ, Coll Lab Med, Key Lab Med Diagnost, Minist Educ, Chongqing 400016, Peoples R China."

来源：BIOSENSORS & BIOELECTRONICS

ESI学科分类：CHEMISTRY

WOS号：WOS:000782658200008

JCR分区：Q1

影响因子：12.6

年份：2022

卷号：206

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：Cascade primer exchange reaction; CRISPR; Cas12a; Ribonuclease H (RNase H); Fluorescence amplification; Ultrasensitive

摘要："Ribonuclease H (RNase H), which plays a vital role in various cellular processes, is to be closely related to the emergence of many diseases. As an essential therapeutic target, it shows great prospects in the development of associated drugs. Herein, a DNA-RNA chimeric hairpin (DR HP) was designed to introduce a new signal amplification strategy based on cascade primer exchange reaction (cPER) and CRISPR/Cas12a system for sensitive and specific analysis of RNase H activity. In the presence of RNase H, the RNA fragment of DR HP was specifically degraded and the blocked primer DNA was released. The process of enzymatic hydrolysis of substrate hairpin and cyclic signal amplification was completed in a one-step method under isothermal conditions, enriching many activator strands to initiate trans-cleavage of CRISPR/Cas system, thereby restoring the fluorescence signal. Under optimized conditions, the developed strategy exhibited a good linear relationship ranging from 0.005 to 0.1U/mL and offered a detection limit of 0.00061U/mL. Moreover, this method was used for RNase H activity assay in complicated human serum and real cell lysates with good stability and repeatability, and was also demonstrated to apply for RNase H inhibitors screening and inhibitory capability assessment. Therefore, the proposed system is a promising platform not only for determination of RNase H activity, but open up new thoughts for the biological enzyme research and inhibitor screening."

基金机构：Key Project of Science and Tech-nology Research Program of Chongqing Education Commission [KJZD-K202000404]; Special Fund Project Key Laboratory of Clinical Laboratory Diagnosis (Ministry of Education)

基金资助正文：Acknowledgements This work was supported by the Key Project of Science and Tech-nology Research Program of Chongqing Education Commission (Grant No. KJZD-K202000404) and the Special Fund Project Key Laboratory of Clinical Laboratory Diagnosis (Ministry of Education) .