Real-time monitoring of the activity and kinetics of T4 polynucleotide kinase by a singly labeled DNA-hairpin smart probe coupled with lambda exonuclease cleavage.

We describe a novel method for real-time monitoring of the activity and kinetics of T4 polynucleotide kinase (PNK) by use of a singly fluorophore-labeled DNA-hairpin smart probe (SP) coupled with lambda exonuclease (lambda exo) cleavage. The method was performed in a sealed reaction tube and offered more sensitive, fast, high-throughput, and cost-effective detection. The SP was designed with a fluorophore at the 3'-end, and the fluorescence was quenched by a GGG-triplet at the 5'-end without any other additional quenchers. The 5'-hydroxyl group of the SP was phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product was promptly cleaved by lambda exo, which caused significant enhancement of fluorescence. A fast and accurate method for assaying the kinase activity of T4 PNK was developed with a wide linear detection range from 0.022 to 5.6 nM s(-1). The phosphorylation reaction was monitored at varying substrate concentrations at the molecular level, and K(m), V(max), and K(cat) values were all calculated. Furthermore, the effects of ATP concentration and salts were investigated. The developed method can be easily adapted to the detection of many other nucleic acid enzymes and may find widespread applications.