Analytical Biochemistry 464 (2014) 63–69

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Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification Mangjuan Tao, Jing Zhang, Yan Jin ⇑, Baoxin Li Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, and Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China

a r t i c l e

i n f o

Article history: Received 17 May 2014 Received in revised form 9 July 2014 Accepted 11 July 2014 Available online 21 July 2014 Keywords: DNA phosphorylation Polynucleotide kinase activity Signal amplification

a b s t r a c t DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acids. Here, an enzyme-assisted amplification strategy was developed for the ultrasensitive monitoring activity and inhibition of T4 PNK. A hairpin oligonucleotide (hpDNA) was designed as a probe whose stem can be degraded from the 50 to 30 direction by lambda exonuclease (k exo) when its 50 end is phosphorylated by PNK. So, the 30 stem and loop part of hpDNA was released as an initiator strand to open a molecular beacon (MB) that was designed as a fluorescence reporter, leading to a fluorescence restoration. Then, the initiator strand was released again by the nicking endonuclease (Nt.BbvCI) to hybridize with another MB, resulting in a cyclic reaction and accumulation of fluorescence signal. Based on enzyme-assisted amplification, PNK activity can be sensitively and rapidly detected with a detection limit of 1.0  104 U/ml, which is superior to those of most existing approaches. Furthermore, the application of the proposed strategy for screening PNK inhibitors also demonstrated satisfactory results. Therefore, it provided a promising platform for monitoring activity and inhibition of PNK as well as for studying the activity of other nucleases. Ó 2014 Elsevier Inc. All rights reserved.

To date, phosphorylation of the 50 -hydroxyl termini of nucleic acids has gradually aroused extensive attention because it plays a significant role in nucleic acid metabolism, especially in DNA repair during strand interruption and damage [1–4] caused by various exogenous and endogenous agents, including chemical substances [5] and ionizing radiation [6] as well as nucleases. Phosphorylation of the 50 -hydroxyl termini is usually catalyzed by polynucleotide kinase (PNK).1 Therefore, assay of PNK activity is of great importance in biochemical and molecular biology research. Traditionally, radioisotope 32P labeling, polyacrylamide gel electrophoresis (PAGE), and autoradiography technology were widely applied in DNA phosphorylation research and PNK activity assay [7–9]. Nevertheless, several limitations, such as time-consuming procedures, laboriousness, complexity, and high cost, prohibit the wider application of these approaches. During recent years, many efforts have been made in the development of simple and fast methods for assay of PNK activity. Tang and coworkers developed

⇑ Corresponding author. Fax: +86 29 81530727. E-mail address: [email protected] (Y. Jin). Abbreviations used: PNK, polynucleotide kinase; MB, molecular beacon; k exo, lambda exonuclease; FP, fluorescence polarization; Nt.BbvCl, nicking endonuclease; ATP, adenosine triphosphate; ADP, adenosine diphosphate; DTT, dithiothreitol; hpDNA, hairpin oligonucleotide; PKA, protein kinase A. 1

http://dx.doi.org/10.1016/j.ab.2014.07.008 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.

a fluorescence assay based on ligation reaction using molecular beacon (MB) as a connection template and fluorescence reporter to investigate the phosphorylation process of nucleic acids by T4 PNK [10]. Song and Zhao constructed a universal fluorescence assay using a singly labeled DNA hairpin smart probe coupled with lambda exonuclease (k exo) cleavage [11]. The smart probe was designed with a fluorophore at the 50 end, which was quenched by guanosine residues in the complementary stem. Lin and coworkers developed a sensitive and rapid method to study T4 PNK activity and inhibition based on k exo cleavage reaction and a graphene oxide platform [12]. Wu and coworkers successfully screened the DNA phosphorylation process by using graphene oxide as a super quencher [13]. Huang and coworkers reported a fluorescence polarization (FP) nanosensor for studying T4 PNK activity and inhibition by combining k exo cleavage reaction with the strong FP enhancement effect of gold nanoparticles (AuNPs) [14]. Nevertheless, these methods also suffered from several drawbacks such as complex operation procedures, vulnerability to contamination, and low sensitivity. Therefore, it is very urgent to establish a highly sensitive, simple, and effective strategy for detecting T4 PNK activity. Here, a universal strategy based on enzyme-assisted signal amplification has been developed for ultrasensitive monitoring of PNK activity and inhibition. As a commonly used polynucleotide

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kinase, T4 PNK was chosen as a model enzyme. The nicking endonuclease (Nt.BbvCI) and k exo were used to realize signal amplification. Nt.BbvCI had attracted much attention because it can cleave only one strand of the DNA duplex at the specific site to release DNA molecules for further signal amplification [15–17]. Meanwhile, k exo is a highly processive enzyme that catalyzes the removal of 50 mononucleotides from 50 -phosphorylated duplex DNA in the 50 to 30 direction [18–21]. Therefore, 50 -phosphorylated hairpin probe can be degraded by k exo to release an initiator strand that can trigger cyclic hybridization with MB under the aid of Nt.BbvCI, resulting in an accumulation of fluorescence signals. Similarly, PNK inhibitors can be screened by monitoring the changes in fluorescence intensity. So, the enzyme-assisted signal amplification strategy may provide an ultrasensitive, effective, and selective strategy for assay of PNK activity and inhibition.

temperature. Finally, 50 nM MB and 10 U/ml Nt.BbvCI were added to the above mixture and incubated at 37 °C for 30 min prior to the fluorescence measurement. Gel electrophoresis To verify the principle, gel electrophoresis was run. A 12.5% nondenatured polyacrylamide gel analysis of the enzymatic reaction was carried out in 1 TBE (89 mM Tris–boric acid and 1 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0) at 200 V for approximately 30 min. After silver staining, the result was achieved by a Canon camera. Results and discussion Proof of principle

Materials and methods Materials Lambda exonuclease (5000 U/ml) and Nt.BbvCI were obtained from New England Biolabs (UK). T4 polynucleotide kinase (10 U/ll), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and dithiothreitol (DTT) were obtained from Shanghai Sangon Biotechnology (Shanghai, China). All oligonucleotides were synthesized by Sangon Biotechnology (Shanghai, China). The sequences of oligonucleotides used in the assay were 50 -CGGGCTCGCC TAACCTCTAAATTGTCGAGCTGAGGTTAGGCGAGC CCG-30 (hairpin probe, hpDNA) and 50 -(FAM)-CTTGCGCCTAACCTCAGCTCGACCG CAAG-(BHQ)-30 (MB). The underlined letters in both hpDNA and MB indicate the specific recognition sequence of Nt.BbvCI. Tris–HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2 was used to prepare the oligonucleotide stock solutions. All other chemicals were of analytical grade and were used without further purification. Deionized water was obtained through a Milli-Q system. Instruments All fluorescence measurements were made with a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Japan). The vertical electrophoresis system was purchased from Bio-Rad Laboratories (Hercules, CA, USA).

The amplification strategy for PNK activity is depicted in Scheme 1. An hpDNA and an MB were designed as DNA probe and fluorescence reporter, respectively. As shown in Scheme 1, once the 50 -hydroxyl terminal of hpDNA is phosphorylated, its stem will be digested by k exo in the 50 to 30 direction to generate a single-stranded initiator strand (purple line) that is complementary to the loop portion of MB. So, the fluorescence of MB is partially restored. From Fig. 1A, we found that the fluorescence of the MB and MB/hpDNA mixture is very low owing to the hairpin structure bringing the fluorophore close to the quencher (curves a and b). However, the fluorescence of MB had a significant enhancement (44.32 ± 0.32%) when hpDNA was incubated with T4 PNK and k exo (curve d). That is, hpDNA was phosphorylated by T4 PNK and further degraded by k exo. Meanwhile, the loop portion of MB contains a recognition site of Nt.BbvCI. So, in the presence of Nt.BbvCI, the MB strand of the duplex will be specifically cleaved because MB has a recognition site of Nt.BbvCI that cleaves only one strand of DNA of a double-stranded DNA substrate. Then, the initiator strand was released again to hybridize with another MB and triggered the second cyclic reaction, resulting in an accumulation of fluorescence signals. It is clear from Fig. 1A that a 244.10 ± 0.6% increase in fluorescence intensity was obtained in

Amplification assay of PNK activity First, the fluorescence spectra of MB were recorded on a fluorometer (F-7000, Hitachi) with excitation at 480 nm and an emission range from 500 to 600 nm. Then, the phosphorylation and k exo cleavage reactions were performed in 50 mM Tris–HCl buffer containing 10 mM MgCl2, 1 mM ATP, and 5 mM DTT. Next, 50 nM hpDNA was incubated with different concentrations of PNK and 5 U/ml k exo at 37 °C for 30 min. The above reaction system was heated to 75 °C for 10 min and cooled to room temperature. Finally, 50 nM MB and 10 U/ml Nt.BbvCI were added to the above mixture and incubated at 37 °C for 30 min prior to the fluorescence measurement. Kinase inhibitor evaluation The influence of sodium hydrogen phosphate, ammonium sulfate, and ADP on the activity of PNK was carried out. The inhibitors were introduced into the T4 PNK reaction system containing 1 mM ATP, 50 nM hpDNA, 1 U/ml PNK, and 5 U/ml k exo. The mixture solution was incubated at 37 °C for 30 min. Then, the above reaction system was heated to 75 °C for 10 min and cooled to room

Scheme 1. Schematic illustration of enzyme-assisted amplification for monitoring PNK activity and inhibition. The stem part of hpDNA can be degraded from the 50 to 30 direction by k exo only when its 50 end is phosphorylated by PNK. So, the 30 stem and loop part of hpDNA was released as an initiator strand to open an MB, leading to fluorescence restoration. Then, the initiator strand was released again to hybridize with another MB due to the specific cleavage of Nt.BbvCI, resulting in cyclic hybridization and accumulation of fluorescence signal. So, PNK activity can be sensitively and rapidly detected by monitoring the change in fluorescence intensity.

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Fig.1. Fluorescence spectra of MB under different conditions. (A) Proof of principle by monitoring the change in fluorescence response. The concentrations of MB, hpDNA, PNK, k exo, and Nt.BbVCI were 50 nM, 10 nM, 1 U/ml, 5 U/ml, and 10 U/ml, respectively. The phosphorylation and k exo cleavage time was 30 min. The cyclic hybridization time was 30 min. (B) The negative control for studying the reliability of the proposed strategy.

the presence of Nt.BbvCI (curve e). Therefore, each PNK-mediated phosphorylation event can trigger many cycles of fluorescence restoration with the help of k exo and Nt.BbvCI, leading to significant fluorescence amplification for ultrasensitive detection of PNK activity. To further ensure reliability, more control experiments were conducted. First, the influence of nonspecific cleavage was investigated. As shown in Fig. 1B, the fluorescence of the MB/hpDNA mixture remains unchanged on the addition of k exo and inactive PNK. That is, the 50 terminal of hpDNA still retained hydroxylation from when PNK is inactive. Therefore, k exo cannot digest the stem part of hpDNA to release the initiator strand for fluorescence cyclic amplification. A slight increase (8.4%) in fluorescence intensity was observed when the above mixture was further incubated with Nt.BbvCI (Fig. 1B, curve d). It is mainly attributed to the nonspecific cleavage by Nt.BbvCI due to the partial hybridization of hpDNA with MB. However, the nonspecific cleavage by Nt.BbvCI hardly affects the assay of PNK activity because it can be eliminated as background fluorescence. Then, the effect of k exo on the fluorescence was studied. From Fig. 2A, we found that fluorescence intensity of MB/hpDNA is unchanged without k exo even when hpDNA was successfully phosphorylated by PNK. Similarly, the fluorescence intensity is also unchanged in the presence of inactive PNK. It is mainly attributed to the fact that the initiator strand is

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Fig.2. Fluorescence spectra of MB under different conditions. (A) Effect of lambda exnonuclease on the signal transfer. The concentrations of MB, hpDNA, PNK, k exo, Nt.BbVCI, BamHI, and ExoRI were 50 nM, 10 nM, 1 U/ml, 5 U/ml, 10 U/ml, 10 U/ml, and 10 U/ml, respectively. (B) Effect of nicking endonuclease on the signal amplification.

not effectively released. Therefore, fluorescence enhancement is really due to the specific cleavage of phosphorylated hpDNA by k exo. Finally, the key role of Nt.BbvCI in signal amplification was investigated. An obvious increase in fluorescence intensity is observed when hpDNA had been phosphorylated by PNK and further degraded by k exo (Fig. 2B). However, no further enhancement was obtained when Nt.BbvCI was replaced by other endonucleases such as EcoRI and BamHI. All of these results revealed that the enzyme-assisted amplification really and reliably worked as we expected.

Characterization of enzymatic cleavage There are two enzymatic cleavages involved in this enzymeassisted amplification strategy. Gel electrophoresis is a good choice to characterize the biological interaction because it is a method for separation and analysis of macromolecules (DNA, RNA, and proteins) and their fragments based on their size and charge. As shown in Fig. 3, the bands in lanes 1 and 2 correspond to hpDNA and MB, respectively. No new band appeared when hpDNA was incubated with k exo, revealing that hpDNA is not cleaved by k exo because hpDNA is not phosphorylated (lane 3). Contrary to lane 3, a new band corresponding to a relatively short DNA fragment was observed in lane 4 when hpDNA was coincubated with PNK and k exo. That is, hpDNA can be degraded by k exo once it

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increased when the MB concentration continually increased. So, the optimal concentration of MB was chosen as 50 nM. ATP plays an important role in the phosphorylation process, and the absence of ATP will result in the blockage of phosphorylation. Therefore, the effect of the ATP amount was examined. As shown in Fig. S1D, the reaction rate increased with increasing concentrations of ATP and then reached its maximum at the ATP concentration of 1 mM. So, the optimal concentration of ATP was chosen as 1 mM. There are three biological interactions involved in this approach. Therefore, interaction time was a crucial parameter for the enzyme-assisted amplification. As illustrated in Fig. S2A of the supplementary material, the fluorescence intensity gradually increased as reaction time was prolonged and finally reached a maximum at 30 min, suggesting a tendency to complete the phosphorylation and specific cleavage by k exo. Then, the dependence of fluorescence intensity on enzymatic cleavage time of Nt.BbvCI was investigated. An increase in fluorescence intensity was observed as reaction time was prolonged (Fig. S2B). The fluorescence intensity nearly approached the steady state at 30 min, suggesting that the optimal reaction time of Nt.BbvCI is 30 min. Specificity and sensitivity of detection of PNK activity Fig.3. Electrophoresis analysis of reliability of enzyme-aided amplification strategy. Lane M: DNA marker. In lanes 1 to 8, the concentrations of MB, hpDNA, PNK, k exo, and Nt.BbvCI were 2.5 lM, 500 nM, 50 U/ml, 250 U/ml, and 500 U/ml, respectively. The electrophoresis time was 30 min.

is phosphorylated by PNK. On the addition of MB, a band of DNA duplex appeared in lane 5, demonstrating that the initiator strand hybridized with MB. Meanwhile, the band of MB shallowed and the band of initiator strand disappeared. From lanes 4 and 5, we concluded that the initiator strand released by k exo digestion really hybridized with MB. Finally, the effects of Nt.BbvCI and k exo were also verified in lanes 7 and 8. No new band was found when hpDNA and MB was incubated with Nt.BbvCI alone (lane 7). However, new bands of short DNA fragment appeared in the presence of Nt.BbvCI when hpDNA and MB were also incubated with PNK and k exo (lane 6). Similarly, it is clear from lanes 4 and 8 that k exo can cleave hpDNA only when hpDNA is phosphorylated in the presence of PNK. The above discussion demonstrates that the enzymeassisted amplification strategy worked just as we expected. Effect of external factors Lambda exonuclease and Nt.BbvCI play a key role in the sensitive detection of PNK activity because the fluorescence enhancement is ascribed to enzyme-triggered cyclic reaction. First, the effect of k exo concentration on signal amplification was optimized. As shown in Fig. S1A of the online supplementary material, fluorescence intensity increased with increasing k exo concentrations and tended to reach relative maximum fluorescence enhancement when the concentration of k exo was equal to or greater than 5 U/ml. To perform a cost-effective assay, the optimal concentration of k exo is 5 U/ml. Second, the effect of Nt.BbvCI was studied. The fluorescence intensity was greatly enhanced as the concentration of Nt.BbvCI increased from 1 to 30 U/ml (Fig. S1B). However, fluorescence enhancement significantly weakened when the concentration of Nt.BbvCI was greater than 10 U/ml, which was mainly attributed to the increase in background fluorescence induced by nonspecific cleavage of MB by Nt.BbvCI. A maximum signal/noise (S/N) ratio was obtained when the concentration of Nt.BbvCI was 10 U/ml. Therefore, the optimal concentration of Nt.BbvCI is 10 U/ml. Furthermore, as a fluorescence reporter, the amount of MB was optimized. As shown in Fig. S1C, the fluorescence intensity increased with increasing MB concentrations from 15 to 100 nM. However, the background fluorescence also greatly

In this work, we aimed to develop a sensitive and selective method to detect PNK activity in homogeneous solution. To further verify the specificity of PNK detection, the influence of other proteins on the PNK assay was investigated. Four common proteins—lysozyme, EcoRI, BamHI, and IgG—were selected to investigate the specificity of PNK assay. It is obvious in Fig. 4A that only PNK caused a significant increase in fluorescence intensity. That is, lysozyme, EcoRI, BamHI, and IgG had no effect on the detection of PNK activity. High specificity was ensured by combining DNA phosphorylation with specific cleavage of phosphorylated DNA by k exo. HpDNA itself cannot open MB because of the stem– loop structure of hpDNA. Only phosphorylated hpDNA can be specifically digested by k exo to release the initiator strand for triggering cyclic hybridization in the presence of Nt.BbvCI. The influence of other kinases, such as protein kinase A (PKA), was studied. As shown in Fig. 4A, PKA did not affect the assay of PNK activity. So, it is a highly specific method for detection of PNK activity. Furthermore, we used gel electrophoresis to verify the specificity of the amplification strategy. A new band of short DNA fragment appeared only when hpDNA was phosphorylated by PNK. Therefore, the electrophoresis result is consistent with fluorescence (Fig. 4B). PNK is a kind of kinase that regulates the phosphorylation of 50 hydroxyl and is closely related to some vital human diseases such as Werner syndrome, Rothmund–Thomson syndrome, and Bloom’s syndrome [22]. Therefore, the high-sensitivity detection of PNK activity is crucial. The dependence of fluorescence enhancement on PNK activity was studied. A continuous increase in fluorescence intensity is found in Fig. 5 when PNK concentration increased gradually, indicating that more and more phosphorylated hpDNAs were digested to liberate the initiator strand for hybridizing with MB. The fluorescence intensity depends linearly on the concentration of PNK in the range from 0.0005 to 1 U/ml. A detection limit of 1.0  104 U/ml is obtained, which is more sensitive than those in previously reported works (see Table S1 in supplementary material) [20,23–25]. To further verify the enzyme-aided amplification, the fluorescence enhancement in the absence of Nt.BbvCI was studied. Fig. S3 demonstrates that fluorescence intensity was highly related to PNK activity. The fluorescence intensity depends linearly on the concentration of PNK in the range from 0.01 to 1 U/ml. The sensitivity investigation demonstrated once again the role of Nt.BbvCI in signal amplification. Unlike other restriction endonucleases, Nt.BbvCI cleaves only one predetermined DNA

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Fig.4. Influence of common proteins on the specificity of assay of PNK activity. (A) Bar chart of change in fluorescence in the presence of different proteins. The concentration of PNK, BamHI, PKA, and EcoRI was 1 U/ml. The concentration of lysozyme and IgG was 1 lM. (B) Electrophoresis analysis of specificity.

Fig.5. Fluorescence spectra of MB with increasing PNK concentrations. From bottom to top, the concentrations of PNK were 0.0005, 0.005, 0.01, 0.05, 0.1, 0.5, and 1 U/ml, respectively. The inset shows the dependence of fluorescence intensity on the logarithm of PNK concentration.

Fig.6. Inhibition of different inhibitors on activity of T4 PNK. (A) Influence of ADP on activity of PNK. From left to right, the concentrations of ADP were 0, 0.25, 0.5, 2, and 5 mM, respectively. (B) Bar chart of relative activity of PNK in the absence and presence of PNK inhibitors. The concentration of PNK was 1 U/ml. (C) Electrophoresis analysis of the influence of the different inhibitors. The electrophoresis time was 30 min.

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strand of double-stranded DNA. So, MBs that contain a recognition site of Nt.BbvCI can be specifically cleaved to liberate the initiator strand for cyclic hybridization, leading to an accumulation of fluorescence intensity. Based on the above results, a solid conclusion was drawn that it offers a highly sensitive and specific method for detecting PNK activity. PNK activity inhibition evaluation 50 -Polynucleotide kinase can catalyze the phosphorylation of nucleic acids with 50 -hydroxyl termini, which regulates many important cellular events, especially DNA repair during strand damage and interruption. So, the inhibition of nucleotide kinase has a crucial effect on cellular nucleic acid regulation and metabolism [3–5,26]. Here, the influence of three inhibitors on PNK activity was studied. As depicted in Fig. S4 of the supplementary material, the fluorescence intensity decreased gradually with increasing inhibitor concentrations. Under the same concentration, ADP led to more effective inhibition of PNK activity (Fig. 6). The influence of ADP on the activity of PNK is mainly ascribed to the competition reaction between ADP and the 50 -phosphate group of nucleic acid [27]. Meanwhile, the inhibition of PNK activity by sodium hydrogen phosphate (Na2HPO4) and ammonium sulfate [(NH4)2SO4] may be due to the following reasons [28,29]. First, a higher concentration of salt can stabilize the helix structure of double-stranded DNA, which may inhibit the activity of the 50 -hydroxyl group. Second, the conformation of PNK may be affected by a high concentration of salts, leading to the reduction of kinase activity as well as the affinity between kinase and its substrates. We also used gel electrophoresis to verify the influence of the different inhibitors on PNK activity. As shown in Fig. 6C, the activity of PNK was inhibited on the addition of different inhibitors (lanes 5–7) and the inhibition efficiency of ADP is the best. All of these results revealed that it provides a simple method for screening inhibitors of PNK as well as studying PNK activity and inhibition. The kinetics parameter of DNA phosphorylation catalyzed by T4 polynucleotide kinase was studied. It is clear from the Lineweaver– Burk plot in Fig. S5 of the supplementary material that there is a marked linear correlation between 1/V0 (where V0 represents the initial rate) and 1/S (where S represents the substrate concentration), demonstrating that the catalysis reaction of T4 PNK obeys the Michaelis–Menten equation. From the Lineweaver–Burk plot, the important kinetic parameters Km (Michaelis–Menten constant) and Vmax (maximum initial velocity) were both determined. Vmax and Km of T4 PNK were 6.94 nM s1 and 1.1 lM, respectively, which are basically consistent with previously reported values. It has been reported that T4 PNK can phosphorylate a wide variety of DNA and RNA substrates, including single-stranded DNA, double-stranded DNA, and RNA, with the minimum substrate being a 30 -phosphate mononucleoside Therefore, the kinetic parameters vary for different substrates [30–32]. Conclusion An enzyme-assisted amplification strategy has been proposed for highly sensitive and specific monitoring of the activity and inhibition of PNK. High specificity was highlighted by coupling high specificity of hairpin DNA with specific cleavage of phosphorylated DNA by k exo. Meanwhile, PNK activity can be sensitively detected as low as 1.0  104 U/ml by phosphorylation-triggered cyclic amplification, which is much lower than those of most existing methods. More important, inhibition of phosphorylation can be simply studied, which has potential for screening inhibitors of PNK. Furthermore, both assay of PNK activity and inhibition performed in homogeneous solution were finished in 1 h. As a result,

it provides a rapid, simple, highly specific, and sensitive method for the study of kinase activity and inhibition. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21075079 and 21375086) and the Program for New Century Excellent Talents in University (NCET10-0557).

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Highly sensitive fluorescence assay of T4 polynucleotide kinase activity and inhibition via enzyme-assisted signal amplification.

DNA phosphorylation catalyzed by polynucleotide kinase (PNK) is an indispensable process in the repair, replication, and recombination of nucleic acid...
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