Polynucleotide Kinase Research Articles

Mutation in PNKP presenting initially as axonal Charcot-Marie-Tooth disease.

PNKP (polynucleotide kinase 3'-phosphatase, OMIM #605610) product is involved in the repair of strand breaks and base damage in the DNA molecule mainly caused by radical oxygen species. Deleterious variants affecting this gene have been previously associated with microcephaly, epilepsy, and developmental delay.(1) According to a previous report, homozygous loss-of-function substitution in PNKP was associated with cerebellar atrophy, neuropathy, microcephaly, epilepsy, and intellectual disability.(2) Recently, whole-exome sequencing (WES) performed in a cohort of Portuguese families with ataxia with oculomotor apraxia (AOA) disclosed pathogenic variants in PNKP in 11 individuals. Other clinical features in that study included neuropathy, dystonia, cognitive impairment, decreased vibration sense, pyramidal signs, mild elevation in α-fetoprotein, and low levels of albumin. This condition was named AOA type 4 (OMIM #616267), as the phenotype of AOA has been previously associated with 3 other genes: APTX, SETX, and PIK3R5.(3) Altogether, these reports demonstrate the great phenotypic diversity associated with PNKP mutations. In this article, we further enlarge this variability by demonstrating that early-onset axonal sensory-motor neuropathy (or axonal Charcot-Marie-Tooth (CMT) disease) followed years later by ataxia without oculomotor apraxia can be caused by deleterious variants in PNKP. Full consent was obtained from the patient and his parents for this publication. This study was approved by institutional ethics committees.

Crystal Structure of Xanthomonas AvrRxo1-ORF1, a Type III Effector with a Polynucleotide Kinase Domain, and Its Interactor AvrRxo1-ORF2

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) disease on rice plants. Xoc delivers a type III effector AvrRxo1-ORF1 into rice plant cells that can be recognized by disease resistance (R) protein Rxo1, and triggers resistance to BLS disease. However, the mechanism and virulence role of AvrRxo1 is not known. In the genome of Xoc, AvrRxo1-ORF1 is adjacent to another gene AvrRxo1-ORF2, which was predicted to encode a molecular chaperone of AvrRxo1-ORF1. We report the co-purification and crystallization of the AvrRxo1-ORF1:AvrRxo1-ORF2 tetramer complex at 1.64Å resolution. AvrRxo1-ORF1 has a T4 polynucleotide kinase domain, and expression of AvrRxo1-ORF1 suppresses bacterial growth in a manner dependent on the kinase motif. Although AvrRxo1-ORF2 binds AvrRxo1-ORF1, it is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain. AvrRxo1-ORF2 functions to suppress the bacteriostatic activity of AvrRxo1-ORF1 in bacterial cells.

Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer

Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3′-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5′ end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10U mL−1 with a detection limit of 2.1×10−3U mL−1. Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs.

Single prokaryotic cell isolation and total transcript amplification protocol for transcriptomic analysis.

Until recently, transcriptome analyses of single cells have been confined to eukaryotes. The information obtained from single-cell transcripts can provide detailed insight into spatiotemporal gene expression, and it could be even more valuable if expanded to prokaryotic cells. Transcriptome analysis of single prokaryotic cells is a recently developed and powerful tool. Here we describe a procedure that allows amplification of the total transcript of a single prokaryotic cell for in-depth analysis. This is performed by using a laser-capture microdissection instrument for single-cell isolation, followed by reverse transcription via Moloney murine leukemia virus, degradation of chromosomal DNA with McrBC and DpnI restriction enzymes, single-stranded cDNA (ss-cDNA) ligation using T4 polynucleotide kinase and CircLigase, and polymerization of ss-cDNA to double-stranded cDNA (ds-cDNA) by Φ29 polymerase. This procedure takes ∼5 d, and sufficient amounts of ds-cDNA can be obtained from single-cell RNA template for further microarray analysis.

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

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research.

Detection of Bulky Endogenous Oxidative DNA Lesions Derived from 8,5'-Cyclo-2'-deoxyadenosine by ³²P-Postlabeling Assay.

8,5'-Cyclopurine-2'-deoxynucleotides represent a class of oxidative DNA lesions that are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. The 32P-postlabeling assay is an ultrasensitive method that has been extensively used for the detection of carcinogen-DNA adducts in laboratory animal and epidemiological studies. This assay under modified chromatographic conditions is also a suitable and sensitive method for the detection of 8,5'-cyclo-2'-deoxyadenosine (cA). After enzymatic digestion of DNA, and enrichment of the oxidative products from the DNA digest, four dinucleotides containing cA, i.e., Ap-cAp, Cp-cAp, Gp-cAp, and Tp-cAp, are 5'-labeled with [32P]orthophosphate from [γ-32P]ATP, mediated by polynucleotide kinase (PNK). The 32P-labeled cA products are separated by two-dimensional thin-layer chromatography (TLC) and quantified by Instant Imager or by a scintillation counter. The assay only requires 1 to 10 μg of DNA sample and is capable of detecting cA lesions at frequencies as low as 1 in 1010 normal nucleotides. © 2015 by John Wiley & Sons, Inc.

Plasmonic AuNP/g-C3N4 Nanohybrid-based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying DNAzyme-Catalyzed Precipitation Amplification.

A convenient and feasible photoelectrochemical (PEC) sensing platform based on gold nanoparticles-decorated g-C3N4 nanosheets (AuNP/g-C3N4) was designed for highly sensitive monitoring of T4 polynucleotide kinase (PNK) activity, using DNAzyme-mediated catalytic precipitation amplification. To realize our design, the AuNP/g-C3N4 nanohybrid was initially synthesized through in situ reduction of Au(III) on the g-C3N4 nanosheets, which was utilized for the immobilization of hairpin DNA1 (HP1) on the sensing interface. Thereafter, a target-induced isothermal amplification was automatically carried out on hairpin DNA2 (HP2) in the solution phase through PNK-catalyzed 5'-phosphorylation accompanying formation of numerous trigger DNA fragments, which could induce generation of hemin/G-quadruplex-based DNAzyme on hairpin DNA1. Subsequently, the DNAzyme could catalyze the 4-chloro-1-naphthol (4-CN) oxidation to produce an insoluble precipitation on the AuNP/g-C3N4 surface, thereby resulting in the local alternation of the photocurrent. Experimental results revealed that introduction of AuNP on the g-C3N4 could cause a ∼100% increase in the photocurrent because of surface plasmon resonance-enhanced light harvesting and separation of photogenerated e-/h+ pairs. Under the optimal conditions, the percentage of photocurrent decrement (ΔI/I0, relative to background signal) increased with the increasing PNK activity in a dynamic working range from 2 to 100 mU mL(-1) with a low detection limit (LOD) of 1.0 mU mL(-1). The inhibition effect of adenosine diphosphate also received a good performance in PNK inhibitor screening research, thereby providing a useful scheme for practical use in quantitative PNK activity assay for life science and biological research.

Label-free detection of T4 DNA ligase and polynucleotide kinase activity based on toehold-mediated strand displacement and split G-quadruplex probes

Herein, an efficient pair of split G-quadruplex probes was selected (S/N=21) to construct a simple, rapid and label-free technique for the detections of T4 DNA ligase and PNK activity. DNA ligase and PNK are engaged in DNA replication and repair, ensuring the genetic stability. The detections of T4 ligase and PNK activity are of great significance in biology and biomedicine. Split G-quadruplex probes are robust label-free biosensing tools with turn-on signal readout. The selected pair of split G-quadruplex probes combined with toehold-mediated strand displacement in this work demonstrated an efficient strategy for monitoring the activities of T4 DNA ligase and PNK with good sensitivity.

Mutations in PNKP Cause Recessive Ataxia with Oculomotor Apraxia Type 4

Hereditary autosomal-recessive cerebellar ataxias are a genetically and clinically heterogeneous group of disorders. We used homozygosity mapping and exome sequencing to study a cohort of nine Portuguese families who were identified during a nationwide, population-based, systematic survey as displaying a consistent phenotype of recessive ataxia with oculomotor apraxia (AOA). The integration of data from these analyses led to the identification of the same homozygous PNKP (polynucleotide kinase 3′-phosphatase) mutation, c.1123G>T (p.Gly375Trp), in three of the studied families. When analyzing this particular gene in the exome sequencing data from the remaining cohort, we identified homozygous or compound-heterozygous mutations in five other families. PNKP is a dual-function enzyme with a key role in different pathways of DNA-damage repair. Mutations in this gene have previously been associated with an autosomal-recessive syndrome characterized by microcephaly; early-onset, intractable seizures; and developmental delay (MCSZ). The finding of PNKP mutations associated with recessive AOA extends the phenotype associated with this gene and identifies a fourth locus that causes AOA. These data confirm that MCSZ and some forms of ataxia share etiological features, most likely reflecting the role of PNKP in DNA-repair mechanisms.

An improved inverse PCR protocol for the generation of T-vectors

T-vectors have been widely used in molecular cloning [1–3]. Compared with common vectors, molecular cloning with T-vectors escapes the restriction endonuclease digestion process, decreases the time of DNA purification and thus is time-saving and cost-effective. There are two ways to produce T-vectors: (i) cutting the target vector with EcoRV and then adding a thymine residue to the 3′-blunt end with TaqDNA polymerase [4]; and (ii) inserting a DNA cassette containing XcmI or AhdI recognition sites at its both ends and then splitting the recombinant vector with them later [5–7]. However, only a small amount of the T-vector can be produced by these methods. What’s more, the residual undigested primary vector may cause false-positive clones. Therefore, T-vectors used in labs are usually bought from biotechnology companies providing with small amounts, thus limiting their application in conventional molecular cloning experiments. Here, we designed an improved inverse PCR protocol for the generation of T-vectors (Fig. 1). Briefly, an inverse PCR was performed with the target vector as a template with a pair of phosphorylated primers, then the PCR products were subject toDpnI treatment, agarose gel purification and further, the terminal deoxynucleotidyl transferase was applied to add a T to the 3′-blunt end of the PCR products which will ligate to the PCR products of the gene of interest with 3′-A overhangs. Afterwards, the ligation mixture was transformed into Escherichia coliDH5α competent cells, and a routine PCR was performed to calculate the positive rate of the transformed clones. The positive clones were further confirmed by DNA sequencing. Finally, we got at least 90% positive clones with our improved protocol. The main materials used in this study includes: the pcDNA3.0 vector, pcDNA3.0-FLAG vector containing human histone H4 cDNA, pcDNA3FLAG-CRBN vector all from our laboratory, PfuUltra II Fusion HS DNA polymerase from Stratagene (Cedar Creek, USA), the E. coli DH5α competent cells, DpnI, premix Taq, and T4 polynucleotide kinase (T4 PNK) fromTaKaRa Biotechnology (Dalian, China), ddTTP fromSigma-Aldrich (St Louis, USA),TaqDNApolymerasewith ThermoPol buffer, terminal deoxynucleotidyl transferase (TdT) from New England Biolabs (NEB; Massachusetts, USA), agarose gel DNA purification kit and PCR purification kit are all from Tiangen Biotech (Beijing, China). The sequence of the primer set for inverse PCR is as follows: forward primer 5′-TTCTATAGTGTCACCTAAATGCTAGA GC-3′, and reverse primer 5′-CCTATAGTGAGTCGTATTAATTT CGATAA-3′. The sequences of the primer pair for FLAGand human histone H4 fusion cDNA are as follows: forward primer 5′-CGGAATT CGCCACCATGGACTACAAGGACGACGA-3′; and reverse primer 5′-C GGGATCCACCGCCGAAACCATAAAGGG-3′; and the sequence of the primer pair for human CRBN cDNA is as follows: forward primer 5′-CCCAAGCTTATGGCCGGCGAAGGAGATC-3′; and reverse primer 5′-CGGGATCCTTACAAGCAAAGTATTACTTTGTC-3′. The T7 and SP6 primer pair was employed in routine PCR to evaluate the positive rate of the transformed clones. The DNA sequence of T7 primer is: 5′-TAATACGACTCACTATAGG-3′. The DNA sequence of SP6 primer is: 5′-ATTTAGGTGACACTATAGAA-3′. DNA sequencing was accomplished with the following primers: forward primer 5′-TTT CCAAAATGTCGTAACAACT-3′, and reverse primer 5′-AGACAATG CGATGCAATTTC-3′. All the above-mentioned primers were synthesized in Sangon Biotech Co., Ltd (Shanghai, China). To determined its usability, the T-vector generated from pcDNA3.0 vector was ligated to the coding sequence of FLAG tag and human histone H4 fusion protein (387 bp) as well as to the cDNA of human CRBN (1329 bp). The positive rate of the transformed clones was evaluated by routine PCR and the rightness of the insertion fragments was confirmed by DNA sequencing. Before the inverse PCR was performed, both the forward and reverse primers were phosphorylated in 20 μl reaction solution containing 5 μl primer (10 μM), 2 μl dNTP (10 mM), 2 μl T4 PNK buffer, 1 μl T4 PNK, and 10 μl distilled water. For inverse PCR, the following reagents were added into a 200 μl PCR tube: 10 ng pcDNA3.0 template plasmid, 400 μM dNTP, 0.8 μM forward and reverse primers respectively, 0.5 μl PfuUltra II Fusion HS DNA polymerase and distilled water was supplemented to 50 μl. Each PCR thermal cycle includes Acta Biochim Biophys Sin, 2015, 47(2), 142–144 doi: 10.1093/abbs/gmu118 Advance Access Publication Date: 23 December 2014 Lab Note

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia type 3 pathogenesis.

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Ataxin-3, DNA damage repair, and SCA3 cerebellar degeneration: on the path to parsimony?

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3’-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

An electrochemical biosensor for the activity assay of polynucleotide kinase and inhibitor screening

A sensitive and selective electrochemical biosensor was fabricated for polynucleotide kinase (PNK) activity assay and inhibitor screening based on phos-tag-biotin mediated double signal amplification.

An amplified fluorescence detection of T4 polynucleotide kinase activity based on coupled exonuclease III reaction and a graphene oxide platform.

A novel amplified fluorescence graphene oxide (GO) sensing platform for sensitive detection of T4 polynucleotide kinase (PNK) activity and inhibition was developed based on the exonuclease III (ExoIII) reaction. The efficient digestion capacity of ExoIII and the super quenching ability of GO both contribute to the detection sensitivity.

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

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.

Phosphorylation-induced hybridization chain reaction on beads: an ultrasensitive flow cytometric assay for the detection of T4 polynucleotide kinase activity.

A versatile flow cytometric bead assay (FCBA) has been developed for the ultrasensitive detection of T4 PNK activity by integrating the advantages of the phosphorylation-induced hybridization chain reaction (HCR) for fluorescence signal amplification and flow cytometry for the robust, sensitive and rapid signal readout of the microbeads (MBs).

Label-free fluorescence light-up detection of T4 polynucleotide kinase activity using the split-to-intact G-quadruplex strategy by ligation-triggered and toehold-mediated strand displacement release.

A label-free, fluorescence light-up detection method for T4 polynucleotide kinase activity has been developed using the split-to-intact G-quadruplex strategy.

A sensitive detection of T4 polynucleotide kinase activity based on β-cyclodextrin polymer enhanced fluorescence combined with an exonuclease reaction.

A strategy for T4 polynucleotide kinase activity detection was proposed based on a β-cyclodextrin polymer (polyβ-CD) and an exonuclease reaction. The fluorescence of pyrene enhanced by more than 10 times in the presence of polyβ-CD, and a simple detection of T4 PNK was achieved with a detection limit of 0.02 units per mL.

One-strand oligonucleotide probe for fluorescent label-free "turn-on" detection of T4 polynucleotide kinase activity and its inhibition.

Thioflavin T (ThT), as one of the most exciting fluorogenic molecules, boasts the "molecular-rotor" ability to induce DNA sequences containing guanine repeats to fold into G-quadruplex structures. It has been demonstrated to sense this change by its remarkable fluorescence enhancement. In this work, taking T4 polynucleotide kinase (PNK) as a model, the ThT/G-quadruplex based platform and λexonuclease (λexo) cleavage reaction were combined to design a label-free "turn-on" strategy for fast, simple and accurate detection of T4 PNK activity and its inhibition. In the presence of T4 PNK, the designed thioflavin T based molecular beacon (TMB) DNA probe could be phosphorylated and then digested by the cleavage of λexo, releasing the G-quartets. These then bound to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation, for the "turn-on" detection of T4 PNK. In comparison to traditional methods, the proposed TMB probe is convenient, requiring no sophisticated labeling and separation processes and displaying high analytical performance. It exhibits a satisfying detection result for the activity of T4 PNK with a low detection limit of 0.001 U mL(-1). This is not only meaningful for further research on disease-related biochemical processes, but also valuable for molecular-target therapies.