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.

Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei.

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5' end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Highly Sensitive Radioactivity-Based DNA 3'-Phosphatase Activity Assay for Polynucleotide Kinase 3'-Phosphatase.

Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such lesions will impede transcription and replication and can lead to various cellular pathologies. Among various "dirty" DNA ends, 3'-phosphate is one of the most abundant lesions generated in the mammalian cells. Polynucleotide kinase 3'-phosphatase (PNKP) is the major DNA end-processing enzyme for resolving 3'-phosphate termini in the mammalian cells, and thus, it is involved in DNA base excision repair (BER), single-strand break repair, and classical nonhomologous end joining (C-NHEJ)-mediated DNA double-strand break (DSB) repair. The 3'-OH ends generated following PNKP-mediated processing of 3'-P are utilized by a DNA polymerase to fill in the gap, and subsequently, the nick is sealed by a DNA ligase to complete the repair process. Here we describe two novel assay systems to detect phosphate release by PNKP's 3'-phosphatase activity and PNKP-mediated in vitro single-strand break repair with minimal repair components (PNKP, DNA polymerase, and DNA ligase) using either purified proteins or cell-free nuclear extracts from mammalian cells/tissues. These assays are highly reproducible and sensitive, and the researchers would be able to detect any significant difference in PNKP's 3'-phosphatase activity as well as PNKP-mediated single-strand break repair activity in diseased mammalian cells/tissues vs normal healthy controls.

DNA protein kinase promotes cellular senescence in dental follicle cells.

OBJECTIVE: Short telomeres and genomic DNA damage are causes of cellular senescence in dental follicle cells (DFCs). DESIGN: This study examined the role of the DNA damage response (DDR) during cellular senescence of DFCs by ß-galactosidase activity and DNA damage by comet assay. Expression of genes/proteins was determined by Western Blots and reverse transcription-quantitative polymerase chain reaction, while glycolysis was enzymatically estimated. Cell cycle stages and reactive oxygen species (ROS) were investigated by flow cytometry. RESULTS: During the induction of cellular senescence gene expression of DDR genes were down-regulated, while DNA double-strand breaks occurred at the same time. Furthermore, inhibition of DNA protein kinase (DNA-PK) reduced senescence and ROS, both of which are associated with cellular senescence. In contrast, while these data suggest that inhibition of DDR is associated with the induction of cellular senescence, inhibition of DNA-PK did not result in renewal of DFCs, as inhibition resulted in typical features of depleted cells such as increased cell size and reduced cell proliferation rate. DNA-PK repression inhibited both osteogenic differentiation potential and glycolysis, which are typical features of cellular exhaustion. Moreover, DNA-PK affects cellular senescence via activation of AKT1 (protein kinase B). CONCLUSION: Our results suggest that DNA-PK promotes cellular senescence, but DFCs may control the induction of cellular senescence via down-regulation of DDR genes. However, we also showed that inhibition of DNA-PK cannot renew senescent DFCs.

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.

Covalent targeting the LAS1-NOL9 axis for selective treatment in NPM1 mutant acute myeloid leukemia.

Patients with NPM1 gene mutation-associated acute myeloid leukemia (AML), particularly those over the age of 60, have no viable targeted therapeutic choices. In this study, we identified HEN-463, a sesquiterpene lactone derivative specific targets AML with this gene mutation. This compound inhibits the interaction of LAS1-NOL9 by covalently binding to the C264 site of the ribosomal biogenesis-related protein LAS1, which translocates the LAS1 to the cytoplasm, thereby inhibiting the maturation of 28 S rRNA. This has a profound effect on the NPM1-MDM2-p53 pathway and ultimately results in the stabilization of p53. Combining this treatment with the XPO1 inhibitor Selinexor (Sel) can ideally preserve the stabilized p53 in the nucleus, considerably enhancing the efficacy of HEN-463 and addressing Sel's drug resistance. Patients with AML over the age of 60 who possess the NPM1 mutation have an unusually elevated level of LAS1, which has a significant impact on their prognosis. In NPM1-mutant AML cells, decreased LAS1 expression promotes proliferation inhibition, apoptosis, cell differentiation, and cell cycle arrest. This suggests that it may be a therapeutic target for this kind of blood cancer, especially in patients over the age of 60.

A label-free fluorescent biosensor for amplified detection of T4 polynucleotide kinase activity based on rolling circle amplification and catalytic hairpin assembly.

T4 polynucleotide kinase (PNK) plays a key role in maintaining genome integrity and repairing DNA damage. In this paper, we proposed a label-free fluorescent biosensor for amplified detection of T4 PNK activity based on rolling circle amplification (RCA) and catalytic hairpin assembly (CHA). Firstly, we designed a padlock probe with a 5'-hydroxyl terminus for phosphorylation reaction, a complementary sequence of the primer for initiating RCA, and a complementary sequence of the trigger for triggering CHA. T4 PNK catalyzed the phosphorylation reaction by adding a phosphate group to the 5'-hydroxyl terminus of padlock probe, generating a phosphorylated padlock probe. Then it hybridized with the primer to generate a circular probe under the action of ligase. Subsequently, the primer initiated an RCA reaction along the circular probe to synthesize a large molecular weight product with repetitive trigger sequences. The triggers then triggered the cyclic assembly reactions between hairpin probe 1 and hairpin probe 2 to generate a large amount of complexes with free G-rich sequences. The free G-rich sequences folded into G-quadruplex structures, and the N-methylmesoporphyrin IXs were inserted into them to produce an amplified fluorescent signal. Benefiting from high amplification efficiency of RCA and CHA, this fluorescent biosensor could detect T4 PNK as low as 6.63 × 10-4 U mL-1, and was successfully applied to detect its activity in HeLa cell lysates. Moreover, this fluorescent biosensor could effectively distinguish T4 PNK from other alternatives and evaluate the inhibitory effect of inhibitor, indicating that it had great potential in drug screening and disease treatment.

Self-Supplying Guide RNA-Mediated CRISPR/Cas12a Fluorescence System for Sensitive Detection of T4 PNKP.

Sensitive detection methods for T4 polynucleotide kinase/phosphatase (T4 PNKPP) are urgently required to obtain information on malignancy and thereby to provide better guidance in PNKP-related diagnostics and drug screening. Although the CRISPR/Cas12a system shows great promise in DNA-based signal amplification protocols, its guide RNAs with small molecular weight often suffer nuclease degradation during storage and utilization, resulting in reduced activation efficiency. Herein, we proposed a self-supplying guide RNA-mediated CRISPR/Cas12a system for the sensitive detection of T4 PNKP in cancer cells, in which multiple copies of guide RNA were generated by in situ transcription. In this assay, T4 PNKP was chosen as a model, and a dsDNA probe with T7 promoter region and the transcription region of guide RNA were involved. Under the action of T4 PNKP, the 5'-hydroxyl group of the dsDNA probe was converted to a phosphate group, which can be recognized and digested by Lambda Exo, resulting in dsDNA hydrolysis. The transcription template was destroyed, which resulted in the failure to generate guide RNA by the transcription pathway. Therefore, the CRISPR/Cas12a system could not be activated to effectively cleavage the F-Q-reporter, and the fluorescence signal was turned off. In the absence of T4 PNKP, the 5'-hydroxyl group of the substrate DNA cannot be digested by Lambda Exo. The intact dsDNA acts as the transcription template to generate a large amount of guide RNA. Finally, the formed Cas12a/gRNA complex triggered the reverse cleavage of Cas12a on the F-Q-reporter, resulting in a "turn-on" fluorescence signal. This strategy displayed sharp sensitivity of T4 PNKP with the limit of detection (LOD) down to 0.0017 mU/mL, which was mainly due to the multiple regulation effect of transcription amplification. In our system, the dsDNA simultaneously serves as the T4 PNKP substrate, transcription template, and Lambda Exo substrate, avoiding the need for multiple probe designs and saving costs. By integrating the target recognition, Lambda Exo activity, and trans-cleavage activity of Cas12a, CRISPR/Cas12a catalyzed the cleavage of fluorescent-labeled short-stranded DNA probes and enabled synergetic signal amplification for sensitive T4 PNKP detection. Furthermore, the T4 PNKP in cancer cells has been evaluated as a powerful tool for biomedical research and clinical diagnosis, proving a good practical application capacity.

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.

Sensitive Autocatalytic Hybridization Circuit for Reliable In Situ Intracellular Polynucleotide Kinase Imaging.

Exploring the characteristic functions of polynucleotide kinase (PNK) could substantially promote the elucidation of PNK-related mechanistic pathways. Yet, the sensitive and reliable detection of intracellular PNK still presents a challenging goal. Herein, we propose a simple autocatalytic hybridization circuit (AHC) for in situ intracellular imaging of PNK with high reliability. The AHC amplifier consists of two mutually activated hybridization chain reaction (HCR) modules for magnified signal transduction. The PNK is transduced into initiator I by phosphorylation and cleavage of mediator Hp. Initiator I activates the initial HCR-1 module, leading to the formation of long dsDNA nanowires that carry numerous initiator T. Then, T-initiated feedback HCR-2 module generates branched products that contain plentiful initiator I, thus realizing an autocatalytic HCR amplification reaction. Simultaneously, the HCR-2 module is also assembled as a versatile signal transduction unit for generating the amplified readout. Based on the mutually sustained accumulation of two initiators for the reciprocal activation of two reaction modules, continuous signal amplification and assembly of high-molecular-weight copolymers endow the AHC system with high sensitivity and robustness for the PNK assay. Moreover, the PNK-sensing AHC system achieves reliable imaging of intracellular PNK, thus showing great potential to decipher the correlation between PNK and related diseases.

The DNA-damage kinase ATR activates the FANCD2-FANCI clamp by priming it for ubiquitination.

DNA interstrand cross-links are tumor-inducing lesions that block DNA replication and transcription. When cross-links are detected at stalled replication forks, ATR kinase phosphorylates FANCI, which stimulates monoubiquitination of the FANCD2-FANCI clamp by the Fanconi anemia core complex. Monoubiquitinated FANCD2-FANCI is locked onto DNA and recruits nucleases that mediate DNA repair. However, it remains unclear how phosphorylation activates this pathway. Here, we report structures of FANCD2-FANCI complexes containing phosphomimetic FANCI. We observe that, unlike wild-type FANCD2-FANCI, the phosphomimetic complex closes around DNA, independent of the Fanconi anemia core complex. The phosphomimetic mutations do not substantially alter DNA binding but instead destabilize the open state of FANCD2-FANCI and alter its conformational dynamics. Overall, our results demonstrate that phosphorylation primes the FANCD2-FANCI clamp for ubiquitination, showing how multiple posttranslational modifications are coordinated to control DNA repair.

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.

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.

An Autocatalytic DNA Circuit Based on Hybridization Chain Assembly for Intracellular Imaging of Polynucleotide Kinase.

Polynucleotide kinase (PNK) plays an essential role in various cellular events by regulating phosphorylation processes, and abnormal homeostasis of PNK could cause many human diseases. Herein, we proposed an autocatalytic hybridization system (AHS) through the elaborate integration of hybridization chain assembly (HCA) and catalytic DNA assembly (CDA) that enables a highly efficient positive feedback amplification. The PNK-targeting AHS biosensor is composed of three modules: a recognition module, an HCA amplification module, and a CDA autocatalytic module. In the presence of PNK, the recognition module could transform the PNK input into an exposed nucleic acid initiator (I). Then the initiator strand I could trigger the autonomous HCA process in the amplification module, and the resulted HCA products could reassemble the split CDA trigger strand T, subsequently inducing the CDA process in the autocatalytic module to form abundant DNA duplex products. Consequently, the embedded initiator strand I was liberated from the CDA duplex product to autonomously trigger the new rounds of HCA circuit. The rational integration and cooperative cross-activation between the HCA and CDA module could prominently accelerate the reaction and realize the exponential amplification efficiency by initiator regeneration. As a result, the self-sustainable AHS amplifier could implement the sensitive detection of PNK in vitro and in biological samples and further fulfill accurate monitoring of the intracellular PNK activity and the effective screening of PNK inhibitors. This work paves a way for exploiting highly efficient artificial DNA circuits to analyze low-abundance biomarkers, holding great potential in biochemical research and clinical diagnosis.

Black Phosphorus Quantum Dots Enhance the Radiosensitivity of Human Renal Cell Carcinoma Cells through Inhibition of DNA-PKcs Kinase.

Renal cell carcinoma (RCC) is one of the most aggressive urological malignancies and has a poor prognosis, especially in patients with metastasis. Although RCC is traditionally considered to be radioresistant, radiotherapy (RT) is still a common treatment for palliative management of metastatic RCC. Novel approaches are urgently needed to overcome radioresistance of RCC. Black phosphorus quantum dots (BPQDs) have recently received great attention due to their unique physicochemical properties and good biocompatibility. In the present study, we found that BPQDs enhance ionizing radiation (IR)-induced apoptotic cell death of RCC cells. BPQDs treatment significantly increases IR-induced DNA double-strand breaks (DSBs), as indicated by the neutral comet assay and the DSBs biomarkers γH2AX and 53BP1. Mechanistically, BPQDs can interact with purified DNA-protein kinase catalytic subunit (DNA-PKcs) and promote its kinase activity in vitro. BPQDs impair the autophosphorylation of DNA-PKcs at S2056, and this site phosphorylation is essential for efficient DNA DSBs repair and the release of DNA-PKcs from the damage sites. Consistent with this, BPQDs suppress nonhomologous end-joining (NHEJ) repair and lead to sustained high levels of autophosphorylated DNA-PKcs on the damaged sites. Moreover, animal experiments indicate that the combined approach with both BPQDs and IR displays better efficacy than monotreatment. These findings demonstrate that BPQDs have potential applications in radiosensitizing RCC cells.

Real-Time Investigation of Intracellular Polynucleotide Kinase Using a Cascaded Amplification Circuit.

Polynucleotide kinase (PNK) shows an in-depth correlationship with DNA repair and metabolism processes. The in situ visualization of intracellular PNK revealed an extremely biological significance in supplementing reliable and quantitative information on its spatiotemporal distribution in live cells. Herein, we developed a versatile cascaded DNA amplification circuit through the integration of catalytic DNA assembly and hybridization chain reaction circuits and realized the accurate evaluation of intracellular PNK activity via the Förster resonance energy transfer (FRET) principle. Initially, without PNK, trigger T was firmly caged in the PNK-recognizing hairpin HT, resulting in no disturbance of the concatenated circuit. However, with the introduction of PNK, the 5'-OH terminal of PNK-addressing HT was phosphorylated, then the phosphorylated HT could be subsequently digested by λ exonuclease (λ Exo) to produce trigger T of the cascaded DNA circuit. As a result, the integrated circuit was stimulated to produce an amplified FRET signal for quantitatively monitoring the activity of PNK. Due to the λ Exo-specific digestion of 5'-phosphate DNA and the high signal gain of the cascade circuit, our proposed strategy enables the sensitive analysis of PNK activity in vitro and in complex biological samples. Furthermore, our PNK-sensing platform was extensively explored in HeLa cells for realizing reliable intracellular PNK imaging and thus showed high potential in the future diagnosis and treatment of kinase-related diseases.

Discovery of potential inhibitors targeting the kinase domain of polynucleotide kinase/phosphatase (PNKP): Homology modeling, virtual screening based on multiple conformations, and molecular dynamics simulation.

In recent years, the level of interest has been increased in developing the DNA-repair inhibitors, to enhance the cytotoxic effects in the treatment of cancers. Polynucleotide kinase/phosphatase (PNKP) is a critical human DNA repair enzyme that repairs DNA strand breaks by catalyzing the restoration of 5'-phosphate and 3'-hydroxyl termini that are required for subsequent processing by DNA ligases and polymerases. PNKP is the only protein that repairs the 3'-hydroxyl group and 5'-phosphate group, which depicts PNKP as a potential therapeutic target. Besides, PNKP is the only DNA-repair enzyme that contains the 5'-kinase activity, therefore, targeting this kinase domain would motivate the development of novel PNKP-specific inhibitors. However, there are neither crystal structures of human PNKP nor the kinase inhibitors reported so far. Thus, in this present study, a sequential molecular docking-based virtual screening with multiple PNKP conformations integrating homology modeling, molecular dynamics simulation, and binding free energy calculation was developed to discover novel PNKP kinase inhibitors, and the top-scored molecule was finally submitted to molecular dynamics simulation to reveal the binding mechanism between the inhibitor and PNKP. Taken together, the current study could provide some guidance for the molecular docking based-virtual screening of novel PNKP kinase inhibitors.

A ligation-driven CRISPR-Cas biosensing platform for non-nucleic acid target detections.

Herein, we describe a CRISPR-Cas12a sensing platform activated by a DNA ligation reaction for the sensitive detection of non-nucleic acid targets, including NAD+, ATP and polynucleotide kinase (PNK). In this design, the DNA ligation reaction triggered by these biomolecules generates DNA duplexes, which can activate the nuclease activity of Cas12a to produce amplified fluorescence signals. As a result, this work provides an alternative strategy to expand the applicability of the CRISPR-Cas system into the detection of non-nucleic acid biomolecules.

Zirconium ion-mediated assembly of a single quantum dot-based nanosensor for kinase assay.

We demonstrate the zirconium ion-mediated assembly of a single quantum dot (QD)-based nanosensor for accurate detection of protein kinases (PKA) and polynucleotide kinases (PNK). This nanosensor is very sensitive with a detection limit of 8.82 × 10-4 U mL-1 for PKA and 1.40 × 10-5 U mL-1 for PNK. Moreover, it can be used to analyze the enzyme kinetic parameters and screen the inhibitors of PKA and PNK, with potential applications in drug discovery and clinical diagnosis.

Hierarchical Metal-Organic Framework-Confined CsPbBr3 Quantum Dots and Aminated Carbon Dots: A New Self-Sustaining Suprastructure for Electrochemiluminescence Bioanalysis.

All-inorganic lead halide perovskites have become promising alternatives to traditional semiconductor electrochemiluminescence (ECL) emitters because of their appealing optoelectronic attributes, but major challenges remain in improving their stability and enhancing charge injection/transfer capacities. Herein, a self-sustaining suprastructure was constructed by successively loading aminated carbon dots (NCDs) and CsPbBr3 perovskite quantum dots (PeQDs) in situ into hierarchical zeolite imidazole framework-8 (HZIF-8). The elaborated architecture guarantees not only improved stability via the peripheral HZIF-8 protective barrier but also accelerated charge transport and efficient self-enhanced ECL between PeQDs and the surrounding NCDs in a confined structure. As a result, the ternary nanocomposite is endowed with greatly improved stability and ECL efficiency. Based on this ternary nanocomposite as an electrode substrate, a novel ECL sensing strategy is further proposed for the first time to evaluate T4 polynucleotide kinase activity and screen its inhibitors. This work opens an avenue for the advancement of perovskite-based ECL emitters as well as the development of corresponding applications in the ECL domain.