Development of a tandem signal amplification strategy for label-free sensing polynucleotide kinase activity in cancer cells

Polynucleotide kinase (PNK) involves in various cellular events by regulating phosphorylation processes, and abnormal expression of PNK may induce many human diseases. Herein, we develop a tandem signal amplifi-cation strategy for label-free sensing PNK activity in cancer cells. In the presence of PNK, the hairpin probe is phosphorylated to initiate the ligation reaction with the assistance of T4 DNA ligase. Subsequently, a tandem signal amplification can be activated to generate abundant triggers with the ligated sequence as the template and the corresponding 3 '-OH end of the 5 '- phosphorylated hairpin probe as the primer. Eventually, the addition of SYBR Green II lights up the triggers to produce an enhanced fluorescence signal. Notably, only target PNK can effectively phosphorylate the hairpin probe to initiate the ligation-dependent reaction, endowing this assay with improved specificity. Moreover, the high amplification efficiency of tandem signal amplification endows this assay with enhanced sensitivity. Furthermore, this assay can accurately measure endogenous PNK activity at single-cell level, screen inhibitors, and analyze enzyme kinetic parameters, facilitating PNK-related clinical diagnosis and drug discovery.