T4 PNK Research Articles

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5′-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3′-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3′-monophosphate (3′-AMP) to adenosine-3′,5′-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3′-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 μM, and the turnover number, kcat, was 8 s−1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3′-AMP were determined to be −25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.

2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology.

Alzheimer’s Disease‐impacted Expression of tRNA‐derived Fragments in Body Fluid: Implication for New Disease Biomarkers

AbstractBackgroundAlzheimer’s disease (AD), the most common type of dementia, affects over six million Americans aged 65 years and older. Although numerous research efforts were made on AD, the knowledge of the mechanisms and therapeutic strategies is still very limited. At present, identifying the prodromal stages of AD remains a significant challenge. tRNA‐derived RNA fragments (tRFs), a family of recently discovered small non‐coding RNAs (sncRNAs), express high in both human and primate hippocampus. Our recent discovery also revealed that some hippocampus tRF expression is much higher in AD patients compared with age‐matched health individuals.MethodHerein, we further characterized the expression of tRFs in CSF and serum from healthy, mild cognitive impairment (MCI), and AD patients, using a combination of T4 polynucleotide kinase (T4 PNK)‐RNA‐seq and qRT‐PCR specific to tRF validation.ResultOur results indicated tRF5‐ProAGG altered by AD in both CSF and serum samples, and serum AD‐impacted tRF5‐ProAGG showed a correlation to the disease severity. Notably, serum tRF5‐ProAGG showed a change tendency when MCI progresses to AD, suggesting that it is a promising biomarker for AD. We also predicted putative targets of tRF5‐ProAGG using our in‐house computational program and confirmed that some targets, such as SV2B, CDH8, and NEFL, were decreased in AD’s hippocampus. Interestingly, SV2B and tRF5‐ProAGG showed a negative correlation.ConclusiontRF5‐ProAGG shows the potential as an AD biomarker and plays a vital role in disease progression.

An electrochemical biosensor for T4 polynucleotide kinase activity identification according to host-guest recognition among phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite and toluidine blue.

T4 polynucleotide kinase (T4 PNK) helps with DNA recombination and repair. In this work, a phosphate pillar[5]arene@palladium nanoparticles@reduced graphene oxide nanocomposite (PP5@PdNPs@rGO)-based electrochemical biosensor was created to identify T4 PNK activities. The PP5 used to complex toluidine blue (TB) guest molecules is water-soluble. With T4 PNK and ATP, the substrate DNA, which included a 5'-hydroxyl group, initially self-assembled over the gold electrode surface by chemical adsorption of the thiol units. Strong phosphate-Zr4+-phosphate chemistry allowed Zr4+ to act as a bridge between phosphorylated DNA and PP5@PdNPs@rGO. Through a supramolecular host-guest recognition connection, TB molecules were able to penetrate the PP5 cavity, where they produced a stronger electrochemical response. With a 5 × 10-7 U mL-1 detection limit, the electrochemical signal is linear in the 10-6 to 1 U mL-1 T4 PNK concentration range. It was also effective in measuring HeLa cell lysate-related PNK activities and screening PNK inhibitors. Nucleotide kinase-target drug development, clinical diagnostics, and screening for inhibitors all stand to benefit greatly from the suggested technology, which offers a unique sensing mechanism for kinase activity measurement.

Large-scale assembly of geometrically diverse metal nanoparticles-based 3D plasmonic DNA nanostructures for SERS detection of PNK in cancer cells

The organization of geometrically diverse metal nanoparticles into a core/satellite structure at a large scale is a promising strategy for improve SERS performance due to hot spots localized enrichment and signal increase. However, due to the lack of extensional frames and strong electrostatic repulsion between plasma NPs, the fabrication of such 3D architectures with a high-density periodic hotspot in the focus volume has proven exceedingly difficult. Herein, we demonstrate a facile large-scale assembly of geometrically diverse metal nanoparticles strategy for constructing spatially extended 3D plasmonic nanostructures resembling “signal towers” based on RCA-mediated periodic organization of gold nanospheres (GNS) surrounding gold nanorods (GNRs). Using cancer cell T4 PNK as a model, a padlock probe with 5′- hydroxyl (P-circle) was designed as the T4 PNK substrate. The center Au nanorod was coated with P1 and served as a “pedestal” to allow substantial loading of P-circle after target phosphorylation to initiate the rolling ring amplification reaction (RCA). The resultant DNA nanowire serves as an “antenna” to successively lock numerous Raman reporter P2 (Cy3-P2-SH) through base pairing at regular intervals. Finally, the 3D plasma DNA nanostructures that resemble “signal towers” could be obtained by placing a large number of GNS with a strong affinity for Au–S. The proposed 3D SERS sensor exhibited a sensitivity of LOD as low as 0.274 mU/mL, which was attributed to a substantial electromagnetic field enhancement at the inter-nanoparticle gaps between the adjacent pedestal and antenna. Moreover, by exploiting the synergistic effect of the periodically extended DNA scaffold generated by RCA amplification and the co-assembly of thiol ligand, the loaded GNS can be extended to three-dimensional space, forming a high-density periodic hotspot in the focal volume, thereby ensuring high enhancement and high reproducibility of Raman signals. In addition, this method can be used to quantify T4 PNK in HeLa cells, demonstrating its applicability in diagnosing and estimating PNK-related diseases in complex fluids.

An electrochemical biosensor for detection of T4 polynucleotide kinase activity based on host-guest recognition between phosphate pillar[5]arene and methylene blue

T4 polynucleotide kinase (T4 PNK) is an important DNA repair-related enzyme that plays a crucial role in DNA recombination, replication and damage repair. Herein, an electrochemical biosensor was developed for detection of T4 PNK activity and inhibitor screening based on supramolecular host–guest recognition between phosphate pillar (Dumitrache and McKinnon, 2017) [5] arene (PP5) and methylene blue (MB). The water-soluble PP5 employed as the host for complexation of MB guest molecules. The substrate DNA with 5′-hydroxyl group was first self-assembled on the gold electrode surface through the chemical adsorption of the thiol group, which was phosphorylated in the presence of T4 PNK and adenosine triphosphate (ATP). TiO2 served as a bridge to link phosphorylated DNA and PP5 via the robust phosphate-Ti4+-phosphate chemistry. The immobilized PP5 captured the MB on electrode surface via the supramolecular host–guest recognition interaction, resulting in an enhanced electrochemical response signal. The electrochemical signal is proportional to the T4 PNK concentration in the range of 2 × 10−4 to 5 U mL−1 with a detection limit of 1 × 10−4 U mL−1. It was also successfully used for PNK inhibitor screening and PNK activity assay in HeLa cell lysates sample. The proposed strategy provides a novel sensing platform for kinase activity assay and inhibitor screening, holding a great potential in clinical diagnostics, inhibitor screening, and nucleotide kinase-target drug discovery.

Red fluorescence-emitting copper nanoparticles-assisted label-free bioprobe coupled with CRISPR/Cas12a for sensitive detection of T4 polynucleotide kinase

With CRISPR/Cas12a and poly T-templated copper nanoparticles (CuNPs), we built an efficient and label-free fluorescent biosensor for determinating T4 polynucleotide kinase (T4 PNK). Single-stranded DNA (ssDNA) of poly T containing 40 bases (T40) was employed as the template for the synthesis of CuNPs. As a result of the Cas12a/crRNA combining with dsDNA, the Cas12a was activated and capable of cleaving T40 and prevented the synthesis of CuNPs. However, the target T4 PNK could phosphorylate the 5′-OH termini of dsDNA, then dsDNA was further degraded by λ exo, so the Cas12a could not be activated. Therefore, the CuNPs were formed, achieving the detection of T4 PNK in an isothermal and label-free way. As expected, the sensing platform with a low limit of detection of 0.089 U mL−1 was proved to be effective for screening and evaluating the inhibitors of T4 PNK. It is very economic to use T40 as the substrate of Cas12a instead of ssDNA probes labeled with fluorophores and quenchers. And the CuNPs can be synthesized in only five minutes. Considering the flexibility of CRISPR/Cas12a, this universal signal output strategy offers great potential to broaden its application scope for other targets assay, such as DNA, proteins, and small molecules.

Probe-labeled electrochemical approach for highly selective detection of 5-carboxycytosine in DNA

5-carboxycytosine (5caC) plays a critical role as an intermediate form in DNA methylation and demethylation processes. Its distribution and quantity significantly influence the dynamic equilibrium of these processes, thereby impacting the normal physiological activities of organisms. However, the analysis of 5caC presents a significant challenge due to its low abundance in the genome, making it almost undetectable in most tissues. In response to this challenge, we propose a selective method for 5caC detection using differential pulse voltammetry (DPV) at glassy carbon electrode (GCE), hinging on probe labeling. The probe molecule Biotin LC-Hydrazide was introduced into the target base and the labeled DNA was immobilized onto the electrode surface with the help of T4 polynucleotide kinase (T4 PNK). Leveraging the precise and efficient recognition of streptavidin and biotin, streptavidin-horseradish peroxidase (SA-HRP) on the surface of the electrode catalyzed a redox reaction involving hydroquinone and hydrogen peroxide, resulting in an amplified current signal. This procedure allowed us to quantitatively detect 5caC based on variations in current signals. This method demonstrated good linearity ranging from 0.01 to 100 nM with a detection limit as low as 7.9 pM. We have successfully applied it to evaluate the 5caC levels in complex biological samples. The probe labeling contributes to a high selectivity for 5caC detection, while the sulfhydryl modification via T4 PNK efficiently circumvents the limitation of specific sequences. Encouragingly, no reports have been made about electrochemical methods for detecting 5caC in DNA, suggesting that our method offers a promising alternative for 5caC detection in clinical samples.

Kanamycin triggered nanozyme for homogeneous and amplified colorimetric detection of T4 polynucleotide kinase

It is of significance to develop efficient methods for detecting the activity of T4 polynucleotide kinase (T4 PNK) due to its essential role in the modulation of different life activities. In this work, we constructed a novel nanozyme using Kanamycin (KANA) as a trigger for the [Fe(CN)6]3- coordinated Cu2(OH)3NO3 (Cu2(OH)3NO3/[Fe(CN)6]3-) nanorods, and designed an amplified colorimetric method to detect T4 PNK. That was, the free KANA efficiently triggered the peroxidase-like activity of Cu2(OH)3NO3/[Fe(CN)6]3-, while the bound KANA by its aptamer lost the stimulative capability for the nanomaterials. On the basis of the bioreaction regulated generation of the KANA aptamer, a highly sensitive colorimetric assay aided by the rolling circle amplification (RCA) reaction for the detection of T4 PNK was realized. Results showed that this assay can detect T4 PNK from 1.0 × 10−3 to 10.0 U/mL, with a limit of detection (LOD) of 1.42 × 10−4 U/mL. The assay also showed acceptable performance in the detection of T4 PNK in serum samples. In addition to the satisfactory sensitivity and selectivity, the displayed T4 PNK assay also presented merits of operational convenience, without labeling or immobilization process and did not require costly instrument. It is expected that the KANA as a stimulator would have extended biosensing applications by coupling various bioreactions that can produce the KANA aptamer.

A label-free T4 polynucleotide kinase fluorescence sensor based on split dimeric G-quadruplex and ligation-induced dimeric G-quadruplex/thioflavin T conformation

The phosphorylation process of DNA by T4 polynucleotide kinase (T4 PNK) plays a crucial role in DNA recombination, DNA replication, and DNA repair. Traditional monomeric G-quadruplex (G4) systems are always activated by single cation such as K+ or Na+. The conformation transformation caused by the coexistence of multiple cations may interfere with the signal readout and limit their applications in physiological system. In view of the stability of dimeric G4 in multiple cation solution, we reported a label-free T4 PNK fluorescence sensor based on split dimeric G4 and ligation-induced dimeric G4/thioflavin T (ThT) conformation. The dimeric G4 was divided into two independent pieces of one normal monomeric G4 and the other monomeric G4 fragment phosphorylated by T4 PNK in order to decrease the background signal. With the introduction of template DNA, DNA ligase, and invasive DNA, the dimeric G4 could be generated and liberated to combine with ThT to show obvious fluorescence signal. Using our strategy, the linear range from 0.005 to 0.5 U mL-1, and the detection limit of 0.0021 U mL-1 could be achieved without the consideration of interference caused by the coexistence of multiple cations. Additionally, research in real sample determination and inhibition effect investigations indicated its further potential application value in biochemical process research and clinic diagnostics.

Construction of a simple, localized and homogeneous fluorescence detection platform for T4 PNK activity based on tetrahedral DNA nanostructure-mediated primer exchange reaction

T4 polynucleotide kinase (T4 PNK), which acts as a bifunctional enzyme, is widely involved in important phosphorylation and dephosphorylation processes. Sensitive and specific detection of T4 PNK activity will help to elucidate its critical importance in disease diagnosis and treatment. The conventional detection method uses hairpin or single-strand primers modified with phosphorylation group as the substrates for T4 PNK hydrolysis. However, considering to the large spatial distances and disordered reaction orientation among the substrates and enzymes, the efficiency and sensitivity of the method are somewhat limited. Here, we propose a simple and sensitive signal amplification strategy, integrating the tetrahedral DNA nanostructure with primer exchange reaction (TDN-PER), which ensures different reaction direction and improves the local reaction concentrations. Compared to dispersed single PER, localized TDN-PER provides more primers for initiating signal amplification. Benefiting from the unique structural transformation from dispersion to localization, this strategy provided ultrahigh sensitivity, achieving a detection limit of 1.8 × 10−5 U/mL, while maintaining excellent accuracy and specificity in complex human serum and cell lysates environments. The method successfully evaluated the inhibitory effects of (NH4)2SO4 and Na2HPO4 on T4 PNK activity. Finally, the practical application capability of this strategy was verified in the detection of cell-free extracts, providing a promising analytical platform for T4 PNK-related biological and medical research.

Recent Developments in G-Quadruplex Binding Ligands and Specific Beacons on Smart Fluorescent Sensor for Targeting Metal Ions and Biological Analytes.

The G-quadruplex structure is crucial in several biological processes, including DNA replication, transcription, and genomic maintenance. G-quadruplex-based fluorescent probes have recently gained popularity because of their ease of use, low cost, excellent selectivity, and sensitivity. This review summarizes the latest applications of G-quadruplex structures as detectors of genome-wide, enantioselective catalysts, disease therapeutics, promising drug targets, and smart fluorescence probes. In every section, sensing of G-quadruplex and employing G4 for the detection of other analytes were introduced, respectively. Since the discovery of the G-quadruplex structure, several studies have been conducted to investigate its conformations, biological potential, stability, reactivity, selectivity for chemical modification, and optical properties. The formation mechanism and advancements for detecting different metal ions (Na+, K+, Ag+, Tl+, Cu+/Cu2+, Hg2+, and Pb2+) and biomolecules (AMP, ATP, DNA/RNA, microRNA, thrombin, T4 PNK, RNase H, ALP, CEA, lipocalin 1, and UDG) using fluorescent sensors based on G-quadruplex modification, such as dye labels, artificial nucleobase moieties, dye complexes, intercalating dyes, and bioconjugated nanomaterials (AgNCs, GO, QDs, CDs, and MOF) is described herein. To investigate these extremely efficient responsive agents for diagnostic and therapeutic applications in medicine, fluorescence sensors based on G-quadruplexes have also been employed as a quantitative visualization technique.

Ultra-specific fluorescence detection of DNA modifying enzymes by dissipation system

Abnormal expression of DNA modifying enzymes (DMEs) is linked to a variety of diseases including cancers. It is desirable to develop accurate methods for DME detection. However, the substrate-based probe for target DMEs is disturbed by various non-target DMEs that have similar activity resulting in a loss of specificity. Here we utilized dissipative DNA networks to develop an ultra-specific fluorescence assay for DME, absolutely distinguishing between target and non-target enzymes. Unlike the conventional sensors in which the discrimination of target and non-target relies on signal intensity, in our system, target DMEs exhibit featured fluorescence oscillatory signals, while non-target DMEs show irreversible ‘one-way’ fluorescence increase. These dissipation-enabled probes (DEPs) exhibit excellent generality for various types of DMEs including DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase (T4 PNK), and methyltransferase (Dam). DEPs provide a novel quantification mode based on area under curve which is more robust than those intensity-based quantifications. The detection limits of APE1, T4 PNK, and Dam reach 0.025 U/mL, 0.44 U/mL, and 0.113 U/mL, respectively. DEPs can accurately identify their corresponding DMEs with excellent specificity in cell extracts. Fluorescence sensors based on DEPs herein represent a conceptually new class of methods for enzyme detection, which can be easily adapted to other sensing platforms such as electrochemical sensors.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase (T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle, the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope (TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4 PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

Ultrasensitive sensing of T4 PNK phosphatase activity through establishing a novel transcription-based signal amplification platform

Signal amplification plays a significant role in nucleic acid analysis, especially in trace nucleic acid detection. Transcription-based signal amplification is a very important component in the field of signal amplification, and has received extensive attention depending on outstanding cyclic transcription ability. However, the low efficiency of transcription caused by limited template length usually restricts their further applications significantly. Herein, we developed a transcription-based signal amplification method with high sensitivity and selectivity. The approach we proposed couples the advantageous features of long template synthesis from phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) with self-amplification from cell-free transcription system. The results demonstrate that this approach can be applied to quantify T4 Polynucleotide Kinase (T4 PNK) phosphatase activity in a broad range from 0.00001 to 0.01 U/mL, with a detection limit of 8.1 × 10-6 U/mL. Moreover, this device can be further employed as a superior signal amplification platform for the detection of other analytical targets after moderate modifications.

Highly efficient incorporation of dATP in terminal transferase polymerization forming the ploy (A)n-DITO-1 fluorescent probe sensing terminal transferase and T4 polynucleotide kinase activity

Fluorescent dye DITO-1 has almost no fluorescence in the absence of nucleic acid. G bases in single strand DNA can induce maximum fluorescent enhancement followed by the A bases when it binds the DITO-1. However, the incorporation efficiency of the dATP was higher than dGTP in terminal transferase (TdT) polymerization. As a consequence, ploy (A)n, rather than ploy (G)n via TdT polymerization had the superior photoluminance when it binded DITO-1 fluorescent dye. Here, we developed a high selective and sensitive sensing strategy for assaying TdT and T4 polynucleotide kinase activity (T4 PNK) based on the ploy (A)n-DITO-1 fluorescent probe. An increasing amounts of TdT enzyme could promote the distinct incorporation of dATP on the DNA primer and form poly (A)n ssDNA with a difference in length. A good linear relationship between the ΔF and the concentrations of TdT in a range of 0.2–50 U/mL was obtained and the detection limit was 0.05 U/mL. Based on the experimental results for TdT, we further expanded the application of this method for detection of a series of concentrations of T4 PNK. The ΔF and the logarithm concentrations of T4 PNK in the range of 0.1–10 U/mL showed a good linear response and the detection limit of 0.02 U/mL was obtained. In addition, the detection of T4 PNK in Hela cell lysate was achieved, demonstrating that the proposed method had the potential application in complex system. The ploy (A)n-DITO-1 fluorescent probe had the excellent properties of one-step readout, robustness for target detection in complex system, and easiness operation, and showed the great potential in clinical diagnostics, inhibitor screening, and drug discovery.

Sensitive photoelectrochemical determination of T4 polynucleotide kinase using AuNPs/SnS2/ZnIn2S4 photoactive material and enzymatic reaction-induced DNA structure switch strategy

We report here Au nanoparticles (AuNPs)/SnS2/ZnIn2S4 as a high-performance active material for sensitive photoelectrochemical (PEC) determination of T4 polynucleotide kinase (T4 PNK) using an enzymatic reaction-induced DNA structure switch strategy. To construct the PEC biosensor, a double-stranded DNA probe consisting of a CdS quantum dots (QDs)-labeled single-stranded DNA (sDNA) and its complementary DNA (cDNA) is immobilized on the AuNPs/SnS2/ZnIn2S4 photoactive material. T4 PNK can catalyze the phosphorylation of 5′–OH–terminated sDNA in the double-stranded DNA probe when ATP is present, and λ-exonuclease can catalyze the degradation of the phosphorylated sDNA into small fragments. Then the cDNA forms a hairpin structure so that CdS QDs and AuNPs are in close contact, which can induce exciton-plasma interactions between CdS QDs and AuNPs. The exciton-plasma interactions significantly boost the photocurrent, enabling the “signal on” PEC determination of T4 PNK in the range of 10−4 - 1 U mL−1 with a detection limit of 6 × 10−5 U mL−1. The PEC biosensor can also be used to screen enzyme inhibitors.

Electrochemical detection of T4 polynucleotide kinase activity based on magnetic Fe3O4@TiO2 nanoparticles triggered by a rolling circle amplification strategy.

An ultrasensitive electrochemical detection of the activity and inhibition of T4 polynucleotide kinase (T4 PNK) was developed by using magnetic Fe3O4@TiO2 core-shell nanoparticles, which was triggered by a rolling circle amplification strategy (Fe3O4@TiO2-RCA). We used Fe3O4@TiO2 as a substrate to anchor a DNA primer. DNA S1 with 5'-OH termini was phosphorylated in the presence of T4 PNK and ATP, which was adsorbed on the surface of Fe3O4@TiO2 NPs and served as the primer for subsequent RCA reactions. After adding circular template DNA S2, RCA was initiated in the presence of phi29 DNA polymerase and dNTPs. Then, Fc-labeled DNA S3 (Fc-S3) was hybridized with RCA. The obtained Fe3O4@TiO2-RCA was adsorbed on the surface of a magnetic gold electrode (MGE) by magnetic enrichment, resulting in an enhanced electrochemical signal. The T4 PNK activity can be monitored by measuring the electrochemical signal generated. This electrochemical assay is sensitive to the activity of T4 PNK with a dynamic linear range of 0.00001-20 U/mL and a low detection limit of 3.0×10-6 U/mL. The proposed strategy can be used to screen the T4 PNK inhibitors, so it has great potential in the discovery of nucleotide kinase-target drug and early clinical diagnosis of cancer.

Kinetic Analysis of T4 Polynucleotide Kinase via Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) measures the heat absorbed or released from biochemical processes. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used as a probe to quantify enzyme activity. Kinases are an important class of enzymes that are involved in many types of cellular regulation, but kinetic studies of kinases by ITC have been limited. T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3′‐phosphate group is present. We used ITC to study the T4 PNK‐catalyzed conversion of 3′‐AMP to adenosine‐3′,5′‐bisphosphate. To initiate the reaction, a single injection of 3′‐AMP substrate was made into the ITC cell containing T4 PNK and ATP at pH 7.6 and 30oC. The shape of the resultant Differential Power versus Time curve is determined by the kinetic properties of the enzyme. Integration of the area under the curve yields reaction enthalpy. The apparent ∆H for the reaction was found to vary depending on the type of buffer used. This result was explained by differential protonation of reactants and products at pH 7.6, and indicated the release of approximately 0.6 protons per mole for the forward reaction. Extrapolation was used to estimate an intrinsic (i.e. buffer‐independent) ∆H of ‐25 kJ/mol. The Michaelis‐Menten constant, KM, was 1.3E‐05 M, and the turnover number, kcat, was 8/sec. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3′‐AMP were assessed successfully by single injection ITC analysis. This finding supports the potential to analyze more complex kinase systems using ITC.

A multiple primers-mediated exponential rolling circle amplification strategy for highly sensitive detection of T4 polynucleotide kinase and T4 DNA ligase activity

T4 polynucleotide kinase (T4 PNK) and T4 DNA ligase are two important repair enzymes that play a significant role in maintaining the stability and integrity of genome. Here, we proposed a multiple primers-mediated exponential rolling circle amplification strategy for label-free and highly sensitive fluorescent analysis of T4 PNK and T4 DNA ligase activity. Firstly, we designed a target strand that contained two C-rich sequences, a complementary sequence of the primer, and three cleavage sites of Nt.BbvCI scattered among the three sequences. The 5′-hydroxyl terminus of target strand was catalyzed by T4 PNK to form a phosphorylated target strand. After the phosphorylated target strand was combined with the primer, T4 DNA ligase catalyzed the formation of circular DNA. Then, the primer triggered an exponential rolling circle amplification reaction along the circular DNA template, exponentially generating G-rich sequences. N-methyl mesoporphyrin IX as signal molecules specifically bound to the G-quadruplexs folded by G-rich sequences, generating a detectable fluorescent signal. Benefiting from exponential signal growth, this strategy showed extremely high detection sensitivity with detection limits of 8.11 × 10−5 U mL−1 of T4 PNK and 3.07 × 10−4 U mL−1 of T4 DNA ligase, respectively. In addition, the proposed strategy exhibited excellent inhibitor evaluation effects for two repair enzymes, and could detect T4 PNK in HeLa cell lysate. The obtained results showed that the proposed strategy had excellent application prospects in disease diagnosis and treatment evaluation.