Label-free one-step fluorescent method for the detection of endonuclease activity based on thioflavin T/G-quadruplex

Abstract Endonucleases, one of the basic tool enzymes of modern molecular biology and medical genetics, have also been clarified as the potential targets for antimicrobial and antiviral drugs screening. However, traditional assays to monitor endonuclease activity can be expensive, time-consuming, or laborious. In order to provide new detective platform, we proposed a novel label-free one-step fluorescent method for the detection of endonuclease activity based on cleavage-induced G-quadruplex formation. In this detection system, a simple DNA probe can spontaneously form a duplex structure with recognition sites of EcoRI and prevent the generation of the G-quadruplex. Once EcoRI is present, the recognition sites in the duplex DNA are cleavage, producing a free guanine-rich sequence to form G-quadruplex. When thioflavin T (ThT) is added, a strong fluorescence signal is given by ThT/G-quadruplex, and therefore the EcoRI activity can be detected. After systematic investigation and optimization, this method has gained a sensitive limit of detection at 0.75 U/mL, and a wide detection range between 0.75 U/mL and 120 U/mL. Furthermore, the inhibitory effect of 5-fluorouracil on EcoRI activity was verified and IC50 was calculated. Taken together, these experimental results have proven that this turn-on fluorescent method has considerable analytical performances. As far as we are concerned, this method is the first reported EcoRI assay based on ThT/G-quadruplex, and only one kind of probe and one kind of dye are involved, providing one of the simplest detective strategies on EcoRI. More importantly, the convenience and cost make this one-step method quite attractive for application transformation. Therefore, we hope this method could be a hopeful option for EcoRI activity determination, and further to help monitoring the quality of tool enzymes and promote the development of high-through automatic drug screening system.