Highly sensitive fluorescence detection of target DNA by coupling exonuclease-assisted cascade target recycling and DNAzyme amplification

Because of the intrinsic importance of nucleic acid as bio-targets, the simple and sensitive detection of nucleic acid is very essential for biological studies and medical diagnostics. Herein, a simple, isothermal and highly sensitive fluorescence detection of target DNA was developed with the combination of exonuclease III (Exo III)-assisted cascade target recycling and DNAzyme amplification. A hairpin DNA probe was designed, which contained the 3′-protruding DNA fragment as target recognition unit, the caged DNA fragment in the stem region as target analogue, and the caged 8-17 DNAzyme sequence in the loop region as signal response unit. Upon sensing of target DNA, the 3′-strand of hairpin DNA probe could be stepwise removed by Exo III, accompanied by the releasing of target DNA and autonomous generation of new target analogues for the successive hybridization and cleavage process. Simultaneously, the 8-17 DNAzyme unit could be exponentially released from this hairpin DNA probe and activated for the cyclic cleavage toward the ribonucleotide-containing molecular beacon substrate, inducing a remarkable fluorescence signal amplification for target detection. A low detection limit of 20 fM with an excellent selectivity toward target DNA could be achieved. The developed cascade amplification strategy may be further extended for the detection of a wide spectrum of analytes including protein and biological small molecules by combining DNA aptamer technology.