A label-free, G-quadruplex DNAzyme-based fluorescent probe for signal-amplified DNA detection and turn-on assay of endonuclease

Abstract

A novel G-quadruplex DNAzyme molecular beacon (G-DNAzymeMB) strategy is developed for assays of target DNA and restriction endonuclease. The detection system consists of G-DNAzymeMB strand and a blocker DNA by using the fluorescence of 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) catalyzed by G-DNAzymeMB as a signal reporter. G-DNAzymeMB exhibits peroxidase activity in its free hairpin structure, and forms a catalytically inactive hybrid when hybridized with blocker DNA. Upon displacement of blocker DNA by target DNA or cleavage by restriction endonuclease, G-DNAzymeMB is released and two lateral portions of G-DNAzymeMB form a G-quadruplex structure, resulting in the recovery of catalytic activity which acts as a cofactor to catalyze H2O2-mediated oxidation of H2DCFDA. For DNA detection system, exonuclease III (Exo III)-catalyzed amplification strategy is introduced to improve the sensitivity and target DNA could be detected as low as 0.1pM. With respect to restriction endonuclease detection system, 0.1U/mL EcoRI endonuclease could be detected and this method could be easily transported to other restriction endonuclease analysis by simply changing the recognition sequence. These results demonstrate that the proposed G-DNAzymeMB strategy could be used as a label-free, simple, sensitive and cost-effective approach in analysis of target DNA and restriction endonuclease.