Rapid DNA detection based on self-replicating catalyzed hairpin assembly using nucleotide base analog pyrrolo-deoxycytidine as fluorophore

A rapid signal amplified DNA detection method based on self-replicating catalyzed hairpin assembly (SRCHA) has been proposed. In this SRCHA system, two split target DNA sequences were respectively integrated into hairpin auxiliary probes H1 and H2. H2 was used as fluorescent probe which containing a fluorescent nucleotide base analog pyrrolo-deoxycytidine (P-dC) at the end of the stem. Target DNA can be circularly used in this SRCHA system to form the helix DNA H1-H2 complex, the structure change of H2 will move P-dC from hairpin stem to flexible ssDNA sticky end, leading to fluorescence increase due to the less stacking interaction. Meanwhile, the two spilt target DNA sequence was reunited and the target DNA replicate was obtained, which also can be circularly used as new activator to trigger additional CHA reaction and fluorescence signal was then rapidly and significantly enhanced. This SRCHA system has been successfully employed for DNA detection with picomolar within around 15 min, and provides a potential technology for the real-time rapid bioanalysis.