A Label-Free Bioluminescent Sensor for Real-Time Monitoring Polynucleotide Kinase Activity

Abstract

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.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification

“…The relative activity of T4 PNK was calculated via the ratio of (F(inhibit) À F 0 ) to (F À F 0 ), where F(inhibit) and F were the fluorescence intensity of NMM corresponding to 1 U/mL T4 PNK in the presence and absence of inhibitor, respectively, and F 0 was the fluorescence intensity of NMM/P1P2/hpDNA/PNK mixture. The IC 50 value of ADP and (NH 4 ) 2 SO 4 were evaluated to be 1.45 mM and 11.0 mM, respectively, which are consistent with the reported IC50 values of 1 mM for ADP and 15 mM for (NH 4 ) 2 SO 4 (Liu et al, 2014b(Liu et al, , 2014aDu et al, 2014). The inhibition effect of ADP possibly resulted from the reversible phosphorylation reaction when ADP and 5′-phosphoryl nucleic acids coexisted in the reaction buffer (Hou et al, 2014).…”

Plasmonic AuNP/g-C3N4Nanohybrid-based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying DNAzyme-Catalyzed Precipitation Amplification

“…Using ADP as a model inhibitor toward PNK activity, the validity of the as-prepared AuNP/g-C 3 N 4 nanohybrids in screening the inhibition of PNK was evaluated through adding the different-concentration ADP into 50 mU mL -1 PNK (used as an example) with the above-mentioned system. As shown in Figure 5, the photocurrent responses (∆I) 18 gradually decreased with the increasing ADP level (note: The added ADP has no significant effect on the activity of λ exo [33][34][35][36]. Meanwhile, we also observed that addition of 0.6 mM ADP could cause about 50% decrease in ∆I, whilst the PNK activity was completely inhibited when the concentration of ADP was higher than 2.5 mM, indicating the potential application of the developed assay system for the studies of PNK inhibition.…”

Rolling Circle Amplification as an Efficient Analytical Tool for Rapid Detection of Contaminants in Aqueous Environments

“…In this case, the RCA reaction generates a large amount of pyrophosphate that can be used as an adenyl transferase substrate to produce ATP. Then, firefly luciferase ATP acts as a cofactor to produce a bioluminescent signal [72,73].…”