Label-free detection of EcoRI endonuclease activity via fluorescent DNA logic gate

A novel AND logic gate has been constructed for label-free detection of EcoRI endonuclease activity based on configuration conversion of hairpin probe with EcoRI and K+ as two inputs. An ingenious hairpin DNA (hpDNA) contained a G-rich loop as a quadruplex-forming oligomer and a long stem as the substrate of EcoRI. In the presence of EcoRI, the stem of hpDNA will be specifically cleaved by EcoRI. The shortened stem was opened by K+ to make G-rich DNA fold into a quadruplex. And then G-quadruplex interacts with N-methyl mesoporphyrin IX (NMM) which is a specific G-quadruplex binder, leading to a dramatic increase in fluorescence of NMM. Based on this, EcoRI activity can be easily and sensitively detected. The linear range is from 0.0001 U/μL to 0.03 U/μL, and the detection limit is 6.8 × 10−5 U/μL. The high specificity was ensured due to the target-induced ingenious switch of DNA conformation. Compared with the previous reports, EcoRI activity can be conveniently and specifically detected in homogeneous solution by one step. No sophisticated experimental techniques or chemical modification of DNA is required. Therefore, it offers a cost-effective, highly sensitive and selective fluorescent AND logic gate platform for label-free bioanalysis.