A Label-Free Bioluminescent Sensor for Real-Time Monitoring
Polynucleotide Kinase Activity
Jiao Du
Qinfeng Xu
Xiaoquan Lu
Chun-yang Zhang
10.1021/ac502240c.s001
https://acs.figshare.com/articles/journal_contribution/A_Label_Free_Bioluminescent_Sensor_for_Real_Time_Monitoring_Polynucleotide_Kinase_Activity/2262682
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.
2014-08-19 00:00:00
hairpin DNA probes
ATP
PNK activity assay
monitoring
bioluminescence signal
AMP
bioluminescent sensor