Target-Navigated\nCBT–Cys “Stapling”\nCoupled with CRISPR/Cas12a Amplification for the Photoelectrochemical\nNucleic Acid Assay

Generally, rolling circle amplification\n(RCA) is based\non an enzyme-linked\npadlock extension reaction. Herein, rapid linking that utilizes click\nchemistry for joining sticky ends of DNA molecules was developed.\nThe ends of nucleic acid were modified with 2-cyano-6-aminobenzothiazole\n(CBT) and cystine (Cys–Cys), while glutathione was introduced\nto break the disulfide bond under target navigation and promote the\nlinkage between CBT and Cys at the terminus of the nucleic acid at\npH 7.4. Subsequently, RCA was performed using phi29 polymerase. CRISPR/Cas12a\ncleavage was triggered by the product of RCA amplification. Assisted\nby alkaline phosphatase, the electron exchange process between the\nphotoelectroactive Sb@Co(OH)F nanorod and <i>p</i>-aminophenol\n(<i>p</i>-AP) was collected in the form of photoelectrochemical\n(PEC) signals. Mass spectrometry, gel electrophoresis, and PEC signals\nwere employed to verify the linking process and the RCA coupled with\nCRISPR/Cas12a cleavage amplification. CBT–Cys connection exhibited\na high reaction rate (23.79 M<sup>–1</sup>·s<sup>–1</sup>). This enzyme-free linking process was superior to traditional enzyme\ncatalysis in terms of the reaction environment and linking rate. This\nefficient nonenzymatic joining system holds great potential for constructing\nnonhomologous end joining, modifying DNA with molecules, and facilitating\nnucleic acid–protein modification processes.