Colorimetric Detection of S1 Nuclease Activity using a Hairpin DNA with Split G-Quadruplex

Abstract

Hairpin DNA containing split G-quadruplex at its terminus displayed tagging position-dependent horseradish peroxidase activity. In general, the hairpin DNA coupled with one GGG repeat at its 5’-terminus and three GGG repeats at its 3′-terminus demonstrated strong catalytic activity, while slight activity was observed after exchanging the position of split G-quadruplex sequence. Inspired by these observations, a novel colorimetric biosensor for nuclease activity assay was developed by selecting S1 nuclease as the model. Specifically, S1 nuclease cleaves the split G-quadruplex into mono- or oligonucleotide fragments, thereby suppressing the catalytic activity, accompanied by a color change from blue to colorless. Under optimum conditions, S1 nuclease was determined with a linear range from 1.60 to 17.6 U/mL and a detection limit of 1.458 U/mL. The constructed method was simple and did not require complex sequence design, expensive instrumentation, and probe labeling. Most importantly, this work facilitates a better understanding of the catalytic mechanism of the DNAzyme and provides an example of the practical application of G-quadruplex-based DNAzyme.