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Target-Navigated CBT-Cys "Stapling" Coupled with CRISPR/Cas12a Amplification for the Photoelectrochemical Nucleic Acid Assay

文献类型： 外文期刊

作者： Zheng, Jie 1 ; Wang, Xiaoyu 2 ; Qin, Hongqing 1 ; Hou, Yaxiao 2 ; Yang, Qianqian 3 ; Zhang, Xuzhi 3 ; Hun, Xu 2 ;

作者机构： 1.Qingdao Univ Sci & Technol, Coll Biol Engn, Coll Chem & Mol Engn, Key Lab Opt Elect Sensing & Analyt Chem Life Sci,M, Qingdao 266042, Peoples R China

2.Qingdao Univ Sci & Technol, Coll Chem & Mol Engn, Key Lab Opt Elect Sensing & Analyt Chem Life Sci, Minist Educ,Shandong Key Lab Biochem Anal,Key Lab, Qingdao 266042, Peoples R China

3.Chinese Acad Fishery Sci, Yellow Sea Fisheries Res Inst, State Key Lab Mariculture Biobreeding & Sustainabl, Qingdao 266071, Peoples R China

期刊名称：ANALYTICAL CHEMISTRY （ 影响因子：6.7； 五年影响因子：6.6 ）

ISSN： 0003-2700

年卷期： 2024 年 96 卷 45 期

页码：

收录情况： SCI

摘要： Generally, rolling circle amplification (RCA) is based on an enzyme-linked padlock extension reaction. Herein, rapid linking that utilizes click chemistry for joining sticky ends of DNA molecules was developed. The ends of nucleic acid were modified with 2-cyano-6-aminobenzothiazole (CBT) and cystine (Cys-Cys), while glutathione was introduced to break the disulfide bond under target navigation and promote the linkage between CBT and Cys at the terminus of the nucleic acid at pH 7.4. Subsequently, RCA was performed using phi29 polymerase. CRISPR/Cas12a cleavage was triggered by the product of RCA amplification. Assisted by alkaline phosphatase, the electron exchange process between the photoelectroactive Sb@Co(OH)F nanorod and p-aminophenol (p-AP) was collected in the form of photoelectrochemical (PEC) signals. Mass spectrometry, gel electrophoresis, and PEC signals were employed to verify the linking process and the RCA coupled with CRISPR/Cas12a cleavage amplification. CBT-Cys connection exhibited a high reaction rate (23.79 M-1s(-1)). This enzyme-free linking process was superior to traditional enzyme catalysis in terms of the reaction environment and linking rate. This efficient nonenzymatic joining system holds great potential for constructing nonhomologous end joining, modifying DNA with molecules, and facilitating nucleic acid-protein modification processes.

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