A Primer-RegulatedRolling Circle Amplification (RCA)for Logic-Controlled Multiplexed Enzyme Analysis

DNA-related enzymes are associated with various diseases and have been potential biomarkers for clinical diagnosis. Developing robust and ultrasensitive methods is extremely favorable for the detection of these biomarkers. To this purpose, a primer-regulated rolling circle amplification (RCA) strategy was ingeniously proposed. Briefly, the RCA primer, which was invalidated with 3′-inverted dT (locked state) and unable to initiate an amplification reaction by phi29 DNA polymerase, was embedded with the recognition substrate of the specific enzyme. In the presence of the target, the recognition and cleavage process of the enzyme prompted the release of the 3′-inverted dT and the regeneration of 3′-OH (unlocked state), satisfying the vital prerequisite for RCA. By adopting this programmable and modular design, the recognition substrate can be either single base sites or a specific sequence for different types of enzymes. This also enables us to conduct single or multiple enzyme detection conveniently, relying on a logic-controlled manner including YES, OR, AND, and AND-OR operations. Overall, the proposed strategy is uniquely insightful and provides a universal tool for multiple analyses of diverse DNA-related enzymes.