Polynucleotide kinase (PNK) plays a crucial role in maintaining the
genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of
cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled
DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent
DNA ligation reaction. In this bioluminescent sensor, two hairpin
DNA probes with 5'-protruding terminal are designed as the
phosphate acceptor, and the widely used
phosphate donor of
ATP is substituted by
dCTP. In the absence of PNK, the
ligation reaction cannot be triggered due to the lack of 5'-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-
ligation reaction of the probes is initiated with the release of
AMP, and the subsequent conversion of
AMP to
ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, cost-effective, and free from the autofluorescence interference of
biological matrix, and can be further used for quantitative, kinetic, and inhibition assay.