T4 Polynucleotide Kinase Phosphatase Research Articles

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

A highly sensitive fluorescence method for the detection of T4 polynucleotide kinase phosphatase based on polydopamine nanotubes

T4 polynucleotide kinase phosphatase (T4 PNKP) plays a critical role in various cellular events, such as DNA damage repair, replication, and recombination. Here, we have described a novel biosensor to detect the activity of T4 PNKP based on polydopamine nanotubes (PDANTs) mediated fluorescence resonance energy transfer (FRET). A FAM-labelled (6-carboxyl-fluorescein) hairpin DNA probe with 3′-phosphoryl terminal was designed as the substrate for T4 PNKP. With the addition of PDANTs, the fluorescence of FAM-labelled hairpin DNA probe could be quenched because of the high adsorption of hairpin DNA on PDANTs. When T4 PNKP dephosphorylated the DNA probe, a double-stranded DNA (dsDNA) product was obtained by Klenow fragment polymerase (KF polymerase) on its 3′-hydroxyl terminal, which could retain most of the fluorescence due to the week adsorption of dsDNA on PDANTs. The developed method demonstrates the sensitivity for T4 PNKP assay in the range from 0.05 to 1.5 U mL−1 with the detection limit of 0.005 U mL−1, which endows the proposed strategy with high enough sensitivity for practical detection in cell lysates. With the advantages mentioned above, this novel sensitive strategy has the potential in the study of DNA damage repair mechanisms.

Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/μL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

CRISPR/Cas12a-based dual amplified biosensing system for sensitive and rapid detection of polynucleotide kinase/phosphatase

We reported a CRISPR/Cas-based dual amplified sensing strategy for rapid, sensitive and selective detection of polynucleotide kinase/phosphatase (PNKP), a DNA damage repair-related biological enzyme. In this strategy, a PNKP-triggered nicking enzyme-mediated strand displacement amplification reaction was introduced to enrich the activator DNA strands for CRISPR/Cas. Such an isothermal DNA amplification step, together with subsequent activated CRISPR/Cas-catalyzed cleavage of fluorescent-labeled short-stranded DNA probes, enable synergetic signal amplification for sensitive PNKP detection. The proposed strategy showed a wide linear detection range (more than 3 orders of magnitude ranging from 1× 10−5 to 2.5 × 10−2 U/mL T4 PNKP) and a detection limit as low as 3.3 × 10−6 U/mL. It was successfully used for the PNKP activity detection in cell extracts with high fidelity and displayed great potential for enzyme inhibitor screening and inhibitory capability evaluation. This work broadens the applications of CRISPR/Cas12a-based sensors to biological enzymes and provides a way to improve the sensitivity by introducing an isothermal signal amplification step. Such an isothermal DNA amplification-CRISPR/Cas-combined biosensor design concept might expand CRISPR/Cas-based sensing systems and promote their applications in various fields such as disease diagnosis and drug screening.

New method for detection of T4 polynucleotide kinase phosphatase activity through isothermal EXPonential amplification reaction.

As a bifunctional enzyme, T4 polynucleotide kinase phosphatase (T4 PNKP) catalyzes the phosphorylation of 5'-hydroxyl, and also removes the terminal 3'-phosphate group. This is closely related to the restructuring, replication, and damage repair of nucleic acid. In this paper, we describe a new method for the sensitive detection of T4 PNKP activity based on the isothermal EXPonential amplification reaction (EXPAR). T4 PNKP can be linearly assayed in the range from 0.001 to 0.01 U mL-1 with a detection limit of 7.9 × 10-4 U mL-1. Moreover, the method exhibits high specificity and sensitivity and can be applied in the enzyme analysis of complex serum samples. In view of its simplicity and moderate experimental conditions, the method may suitable for use in a commercial kit for the analysis of T4 PNKP activity.

Determination of the activity of T4 polynucleotide kinase phosphatase by exploiting the sequence-dependent fluorescence of DNA-templated copper nanoclusters.

A fluorometric method is described for the determination of the activity of the enzyme T4 polynucleotide kinase phosphatase (T4 PNKP). A short 3'-terminus phosphorylated DNA strand is hybridized with a long DNA strand to produce a partially double-stranded DNA (dsDNA) substrate. On addition of T4 PNKP, the substrate is dephosphorylated to generate the long dsDNA, and then the long dsDNA acted as a template for synthesizing copper nanoclusters (CuNCs). The dsDNA-templated CuNCs display fluorescence with excitation/emission peak wavelengths of 340/570nm. The fluorescence is DNA sequence-dependent. A DNA substrate was designed to enhance fluorescence and to reduce the background in order to improve the sensitivity of the assay. The assay has an analytical range that extends from 0.07UmL-1 to 15UmL-1 and a detection limit of 0.06UmL-1. Graphical abstract The sequence-dependent fluorescence of DNA-templated copper nanoclusters, which are in-situ synthesized through the reduction of CuSO4 by ascorbate (Vc) in the presence of dsDNA template, is utilized to obtain the method for sensitive detection of T4 PNKP activity with near-zero background.

Label-free fluorescent assay of T4 polynucleotide kinase phosphatase activity based on G-quadruplexe−thioflavin T complex

T4 polynucleotide kinase phosphatase (T4 PNKP) is a bifunctional tool enzyme with 5′-kinase and 3′-phosphatase activities. Considering its important roles in the repair of strand breaks, assay of T4 PNKP activity is of great importance. In this work, we proposed a novel label-free sensing strategy for T4 PNKP activity based on G-quadruplexe−thioflavin T fluorescent indicator. In the assay, we used a single stranded oligonucleotide with 3′-phosphoryl and 5′-phosphoryl ends as enzyme substrate. Upon the addition of T4 PNKP, the 3′-PO3 of the substrate was changed to 3′-OH which initiated the polymerization in the presence of terminal deoxynucleotidyl transferase and G-rich dNTP substrates. The resultant elongated DNA can form G-quadruplex in the inducement of K+, resulting in strong fluorescence signal when using thioflavin T as a G-quadruplex−specific light-up fluorescent probe. The detection limit of this method is as low as 0.2U/mL. Additionally, the inhibition of T4 PNKP activity by the inhibitor heparin is demonstrated. This method is easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.

Label-free colorimetric assay for T4 polynucleotide kinase/phosphatase activity and its inhibitors based on G-quadruplex/hemin DNAzyme

Here we report a new approach for label-free colorimetric assay of T4 polynucleotide kinase/phosphatase (PNKP) activity based on G-quadruplex/hemin DNAzyme. In the presence of T4 PNKP, the DNA primer with a 3’-phosphate can be dephosphorylated into a 3’-hydroxyl and initiate a primer elongation reaction to open the hairpin probe, and leading to releasing the G-quartets. Under optimal conditions, the proposed method exhibited a considerable performance with a detection limit of 0.01 U/mL. Furthermore, the present assay can be used to study the potential T4 PNKP inhibitor screening, making it promise to be applied in the fields of drug discovery.

Electrochemical detection of DNA 3′-phosphatases based on surface-extended DNA nanotail strategy

Determination of DNA dephosphorylation is of great value due to its vital role in many cellular processes. Here we report a surface-extended DNA nanotail strategy for simple and ultrasensitive detection of DNA 3′-phosphatases by terminal deoxynucleotidyl transferase (TdT) mediated signal amplification. In this work, DNA probes labeled with thiols at their 5′ terminals and phosphoryls at 3′ terminals are immobilized on gold electrode and are used as substrates for DNA 3′-phosphatases, taking T4 polynucleotide kinase phosphatase (T4PNKP) as an example. T4PNKP can catalyze the dephosphorylation reaction of the substrate DNA, followed by the formation of a long DNA strand by TdT on its 3′ terminal hydroxyl, leading to an evident chronocoulometry signal enhancement. The proposal presents a considerable analytical performance with low detection limit and wide linear range, making it promise to be applied in the fields of DNA dephosphorylation related processes, drug discovery, and clinical diagnostics.

Label-free, isothermal and ultrasensitive electrochemical detection of DNA and DNA 3'-phosphatase using a cascade enzymatic cleavage strategy.

Label-free and ultrasensitive electrochemical assays of target DNA and T4 polynucleotide kinase phosphatase (PNKP) were developed, which took full advantage of three enzymes to realize the signal readout and amplification. It can ultimately achieve the low detection limits of 10 fM and 1 mU mL(-1) for target DNA and PNKP, respectively.

Development of a highly sensitive sensing platform for T4 polynucleotide kinase phosphatase and its inhibitors based on WS2nanosheets

A novel strategy for nucleotide kinase phosphatase activity assay based on WS2nanosheets.

Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification

As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019U/mL. By adding a PNKP-triggered 5′-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

A graphene oxide platform for the assay of DNA 3'-phosphatases and their inhibitors based on hairpin primer and polymerase elongation.

We have developed a label-free, simple and highly sensitive hairpin fluorescent biosensor for the assay of DNA 3'-phosphatases and their inhibitors utilizing a graphene oxide (GO) platform. In this assay, we designed a hairpin primer (HP) with a 3'-phosphoryl end that served as the substrate for DNA 3'-phosphatases. Once the phosphorylated HP was hydrolyzed by DNA 3'-phosphatases, the resulting HP with a 3'-hydroxyl end was immediately elongated to form a long double-strand product by Klenow fragment polymerase (KF polymerase). With SYBR green I (SG) selective staining of the double-helix DNA, a very high fluorescence enhancement was achieved. Furthermore, GO was introduced to quench the fluorescence of the HP without polymerase elongation, thereby further increasing the signal-to-background ratio. The proposed method is simple and convenient, yet still exhibits high sensitivity and selectivity. This method has been successfully applied to detecting the activity of two typical 3'-phosphatases, T4 polynucleotide kinase phosphatase (PNKP) and shrimp alkaline phosphatase (SAP). The effect of their inhibitors has also been investigated. The results revealed that the method allowed a sensitive quantitative assay of T4 PNKP and SAP, with detection limits of 0.07 U mL-1 and 0.003 U mL-1, respectively. The proposed method is anticipated to find applications in the study of DNA damage repair mechanisms.

Mechanism of RNA 2′,3′-cyclic phosphate end healing by T4 polynucleotide kinase–phosphatase

T4 polynucleotide kinase–phosphatase (Pnkp) exemplifies a family of enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The polynucleotide 3′-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3′-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid–base catalyst Asp167. We report that Pnkp also has RNA 2′-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2′,3′-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNAOH, mutant D167N converts RNA > p to RNA 3′-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′-phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNAOH + phosphoaspartyl-Pnkp → Pi + Pnkp.

Bacteriophage T4 polynucleotide kinase triggers degradation of mRNAs

The bacteriophage T4-encoded RegB endoribonuclease is produced during the early stage of phage development and targets mostly (but not exclusively) the Shine-Dalgarno sequences of early genes. In this work, we show that the degradation of RegB-cleaved mRNAs depends on a functional T4 polynucleotide kinase/phosphatase (PNK). The 5'-OH produced by RegB cleavage is phosphorylated by the kinase activity of PNK. This modification allows host RNases G and E, with activity that is strongly stimulated by 5'-monophosphate termini, to attack mRNAs from the 5'-end, causing their destabilization. The PNK-dependent pathway of degradation becomes effective 5 min postinfection, consistent with our finding that several minutes are required for PNK to accumulate after infection. Our work emphasizes the importance of the nature of the 5' terminus for mRNA stability and depicts a pathway of mRNA degradation with 5'- to 3'-polarity in cells devoid of 5'-3' exonucleases. It also ascribes a role for T4 PNK during normal phage development.

Development of a High‐Throughput Screening Platform for DNA 3′‐Phosphatases and Their Inhibitors Based on a Universal Molecular Beacon and Quantitative Real‐time PCR

DNA 3'-phosphatases play a unique role in the repair of strand breaks induced by DNA damaging agents, such as ionizing radiation or oxidative stress. In this paper, we present an efficient detection system for rapid screening of DNA 3'-phosphatases and their inhibitors. A unique template substrate has been designed to hybridize with the universal molecular beacon (U-MB), and the detection process is carried out in a quantitative real-time PCR. The method is successfully applied to monitor the activity and kinetics of two typical 3'-phosphatases, that is, T4 polynucleotide kinase phosphatase (PNKP) and calf intestinal alkaline phosphatase (CIP). The inhibition effect of heparin on T4 PNKP and theophylline on CIP is also quantitatively characterized. The proposed method is demonstrated to be very useful for sensitive, high-throughput, and precise measurement of various 3'-phosphatases and their inhibitors.