Zn²⁺-Ligation DNAzyme-Driven Enzymatic and Nonenzymatic Cascades for the Amplified Detection of DNA

Abstract

A generic fluorescence sensing platform for analyzing DNA by the Zn²⁺-dependent ligation DNAzyme as amplifying biocatalyst is presented. The platform is based on the target DNA induced ligation of two substrate subunits and the subsequent opening of a beacon hairpin probe by the ligated product. The strand displacement of the ligated product by the beacon hairpin is, however, of limited efficiency. Two strategies are implemented to overcome this limitation. By one method, a “helper” nucleic acid sequence is introduced into the system, and this hybridizes with the DNAzyme components and releases the ligated product for opening of the hairpin. By the second method, a nicking enzyme (Nt.BspQI) is added to the system, and this nicks the duplex between the beacon and ligated product while recycling the free ligation product. By combining the two coadded components (“helper” sequence and nicking enzyme), the sensitive detection of the analyte is demonstrated (detection limit, 20 pM). The enzyme-free amplified fluorescence detection of the target DNA is further presented by the Zn²⁺-dependent ligation DNAzyme-driven activation of the Mg²⁺-dependent DNAzyme. According to this method, the Mg²⁺-dependent DNAzyme subunits displace the ligated product, and the resulting assembled DNAzyme cleaves a fluorophore/quencher-modified substrate to yield fluorescence. The method enabled the detection of the target DNA with a detection limit corresponding to 10 pM. The different sensing platforms are implemented to detect the Tay–Sachs genetic disorder mutant