A label-free cyclic assembly of G-quadruplex nanowires for cascade amplification detection of T4 polynucleotide kinase activity and inhibition

Abstract

Several fluorescence methods have been developed for sensitive detection of PNK activity based on signal amplification techniques, but they need fluorescently labeled DNA probes and superabundant assistant enzymes. We have addressed these limitations and report here a label-free and enzyme-free amplification strategy for sensitively and specifically studying PNK activity and inhibition via hybridization chain reaction (HCR). First, the phosphorylation of hairpin DNA H1 by T4 PNK makes it be specifically digested by lambda exonuclease (λ exo) from 5' to 3' direction to generate a single-stranded initiator which can successively open hairpins H2 and H3 to trigger an autonomous assembly of long DNA nanowires. Meanwhile, an intermolecular G-quadruplex is formed between H2 and H3, thereby providing fluorescence enhancement of N-methyl mesoporphyrin IX (NMM) which is a highly quadruplex-selective fluorophore. So, the PNK activity can be facilely and sensitively detected by using NMM as a signal probe which provides a low background signal to improve the overall sensitivity, resulting in the detection limit of 3.37 × 10(-4) U mL(-1). More importantly, its successful application for detecting PNK activity in a complex biological matrix and studying the inhibition effects of PNK inhibitors demonstrated that it provides a promising platform for screening PNK inhibitors as well as detecting PNK activity. Therefore, it is a highly sensitive, specific, reliable and cost-effective strategy which shows great potential for biological process research, drug discovery, and clinical diagnostics.

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ Exonuclease Assistance

“…The IC 50 (half-maximal inhibitory concentration) curves of ADP and (NH 4 ) 2 SO 4 are shown in Figure . The IC 50 values for ADP and (NH 4 ) 2 SO 4 are 1.5 and 12.6 mM, respectively, which are comparable with the literature reported values. − The result confirms that the paper-based fluorescence assay has the potential to screen PNK inhibitors as well as determine corresponding IC 50 values.…”

“…The detection limit of PNK is 0.0001 U mL −1 according to the 3σ rule, which is lower than or comparative with those of previous reports (Table S1). [13][14][15][16][17][18][19][20]39,40 The assay−assay precision coefficient of variation (CV) of the PNK activity detection is below 5.1% in all experimental data sets, indicating that the assay has reasonable reproducibility. In addition, the detection principle is also demonstrated by gel electrophoresis analysis (Figure S6).…”

“…The traditional PNK activity assays including radioactive isotope 32 P-labeling, polyacrylamide gel electrophoresis (PAGE) and autoradiography , have several limitations such as time-consuming, high cost, radioactive contamination, and requirement of skillful technicians. To overcome these disadvantages, varieties of other techniques for the determination of PNK have been developed including colorimetric, , fluorescent, − electrochemical, , chemiluminescent, and so on. Nevertheless, the above techniques are mainly based on solution-phase reaction systems and need complicated instruments, which are not practical for using outside laboratory and cannot meet the demands of point-of-care (POC) test.…”

Detection of T4 Polynucleotide Kinase via Allosteric Aptamer Probe Platform

“…In comparison, the signal change upon addition of T4 PNK was not the decisive factor due to its lesser contribution to S/B ratio (less than two times between the maximum and the minimum). Therefore, SA-6 with the lowest background had the best S/B ratio of 193, which is 1–2 orders of magnitude higher than S/Bs of other fluorescence methods. ,− As a consequence, our method exhibits substantial capability for a low limit of detection, and SA-6 was chosen for use in subsequent experiments.…”

“…T4 polynucleotide kinase (T4 PNK) is a functional enzyme that specifically acts in phosphorylation at the 5′-OH via the transfer of the γ-phosphate from ATP to DNA. This process plays a crucial part in DNA recombination and replication, as well as restoration. − Many researchers have demonstrated that aberrant activity of T4 PNK, which induces abnormal phosphorylation, is related to some serious human diseases such as Rothmund–Thomson Syndrome, Werner Syndrome, and Bloom’s Syndrome. , Accordingly, it is of great importance to establish a highly sensitive detection strategy for T4 PNK, for use in inhibitor screening, clinical diagnostics, and drug discovery.…”