No-nonspecific recognition-based amplification strategy for endonuclease activity screening with dual-color DNA...

The inevitable nonspecific recognition severely restricted widely used nucleic acid amplification strategies, which has become an urgent problem in current scientific research. Herein, we developed a novel no-nonspecific recognition-based amplification strategy to construct dual-color dye loaded nano-clew as ultrabright illuminant for screening endonuclease activity with Escherichia coliRY13 I (EcoR I) as a model, which overcame some major drawbacks such as nonspecific recognition and photobleaching. Typically, the target endonuclease induces cleavage of the customized dumbbell-shape substrate (DSS) to generate two same triggers that can initiate the rolling circle amplification (RCA) to prepare long single-strand DNA (lssDNA), which could self-assemble into irregular DNA nano-clew based on the electrostatic interactions with Mg²⁺ to furtherly capture the donor and accepter fluorophore proximately, constructing the dye loaded nano-clew with dual-color fluorescence (FL) emission to resist photobleaching. Importantly, in absence of EcoR I, even if the DSS could combine with circular template a little, the reaction system performed hardly RCA reaction due to no cohesive terminus, resulting an extremely low background fluorescence signal because of the prevention of nonspecific RCA reaction. As expected, the proposed sensing platform with a low limit of detection (LOD) of 3.4 × 10^-7 U/μL was demonstrated to work well for endonuclease inhibitors screening also. Furthermore, the proposed no-nonspecific recognition strategy could be readily extended to various DNA or RNA enzymes such as DNA methyltransferase, DNA repair-related enzymes and polynucleotide kinase just by simply changing the recognition sequence in the DNA substrate, performing great potential of endonucleases-related clinical diagnosis and drugs discovery.