期刊
ANALYTICAL CHEMISTRY
卷 86, 期 16, 页码 8481-8488出版社
AMER CHEMICAL SOC
DOI: 10.1021/ac502240c
关键词
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资金
- Natural Science Foundation of China [21325523, 21327005, 21175108]
- Program for Changjiang Scholars and Innovative Research Team, Ministry of Education of China [IRTI283]
- Chinese Academy of Sciences
- Program of Innovative Research Group of Gansu Province of China [1210RJIA001]
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.
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