Sensitive and Rapid Screening of T4 Polynucleotide Kinase Activity

Oct 25, 2011 - Zhaoqian Li , Yazhen Li , Mengyu Qi , Shuxian Zhong , Weiping Wang , Ai-Jun Wang , Jianrong Chen ... A tutorial review for employing en...
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Sensitive and Rapid Screening of T4 Polynucleotide Kinase Activity and Inhibition Based on Coupled Exonuclease Reaction and Graphene Oxide Platform Lei Lin, Yang Liu, Xin Zhao, and Jinghong Li* Department of Chemistry, Beijing Key Laboratory for Analytical Methods and Instrumentation, Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing 100084, China

bS Supporting Information ABSTRACT: Phosphorylation of DNA with 50 -hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption. Determination of nucleotide kinase activity and inhibition is under intense development due to its importance in regulating nucleic acid metabolism. Here, by using T4 polynucleotide kinase (PNK) as a model, which plays an essential role in cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, we describe a strategy for simply and accurately determining nucleotide kinase activity and inhibition by means of a coupled λ exonuclease cleavage reaction and graphene oxide (GO) based platform. The dye attached dsDNA preserves most of the fluorescence when mixed with GO. While dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, fluorescence is greatly quenched. Because of the super quenching ability and the high specific surface area of GO, the as-proposed platform presents an excellent performance with wide linear range and low detection limit in the cell extracts environment. Additionally, inhibition effects of adenosine diphosphate, ammonium sulfate, and sodium hydrogen phosphate have also been investigated. The method not only provides a universal platform for monitoring activity and inhibition of nucleotide kinase but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.

hosphorylation of DNA with 50 -hydroxyl termini plays a critical role in a majority of normal cellular events, including DNA recombination, DNA replication, and repair of DNA during strand interruption.14 Efficient and prompt repair of DNA lesions is of fundamental importance in the maintenance of gene integrity and human diseases. Several exogenous and endogenous agents, for instance, chemical substances,5 ionizing radiation,6 as well as nucleases,7 easily induce strand breaks and the following generation of hydroxyl group at the 50 -end of DNA. This fact may have a serious impact on the healing processes for DNA which need the 50 -phosphate terminal and even lead to the failure of DNA repair. In other words, phosphorylation at the 50 termini is usually a prerequisite before broken strands rejoining can be finished. Traditionally, autoradiography, radical isotope 32P-labeling, and PAGE are commonly used for the estimation of DNA phosphorylation kinase activity.813 Unfortunately, several limitations, such as time-consuming, complicated, inefficient and costly, prohibit the broad application of these methods. In these years, a lot of effort has been directed toward the development of fluorescence assays for DNA phosphorylation because fluorescence methods are convenient, high-throughput, cost-effective, and sensitive. Tang et al. described a novel fluorescence assay for screening of the phosphorylation process using a molecular beacon. The method made use of T4 polynucleotide kinase (PNK) and ligation enzyme-coupled reaction, which realized a

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sensitive monitoring of the phosphorylation process.1 Song et al. developed a novel method for real-time investigation of the activity and kinetics of PNK by utilizing fluorophore-labeled DNA-hairpin probes and λ exonuclease coupled cleavage reaction.14 However, design of specific dye labeled DNA hairpin probes is difficult and costly. What’s more, in spite of the development of these fluorescence methods, further improvement of the analytical performances including sensitivity, linear range, etc. is still in urgent need. Accordingly, it remains a challenge in developing accurate, simple, sensitive, and rapid methods for the determination of DNA phosphorylation kinase activity. Recently, graphene, a robust carbon nanomaterial, has received unrivalled attention ascribed to its unique electronic and structure properties.15,16 In addition, the outstanding capability of graphene in fluorescence resonance energy transfer (FRET) has shown that it is a promising candidate for developing fluorescent sensing methods.17 Because of its peculiar electronic properties, graphene is an excellent energy acceptor with super quenching ability, which has been corroborated by photophysical calculations.17 Meanwhile, graphene possesses a high specific surface area, providing a high concentration of biomolecules on it through nonconvalent interactions such as hydrogen bonding or Received: March 7, 2011 Accepted: October 11, 2011 Published: October 25, 2011 8396

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Analytical Chemistry ππ stacking. Applications in endocellular molecular probing,18 DNA detection,19,20 and non-DNA molecular analysis21,22 have proved that graphene can be successfully used to construct various sensing platforms with excellent performance. However, despite its superb properties, the graphene based platform has rarely been applied to detect the activity and inhibition of important enzymes.23,24 T4 polynucleotide kinase (PNK) is a famous member of the 50 -kinase family, which catalyzes the transfer of the γ-phosphate group of nucleoside triphosphate (ATP) to the 50 -hydroxyl end of oligonucleotides or nucleic acids. PNK is essential for cellular nucleic acid metabolism, particularly in the cellular responses to DNA damage, which links with many human disorders as Werner syndrome, Bloom’s syndrome, and Rothmund-Thomson syndrome.25 PNK has also found widespread applications in repair of nucleic acid lesions26 and analysis of DNA adducts27 since its discovery.28 Therefore, accurate monitoring of PNK activity and its potential inhibitors is of considerable importance in nucleic acid metabolism research and moleculartarget therapies. Here, we describe a simple but effective graphene oxide (GO) platform using PNK as a model for nucleotide kinase activity and inhibition analysis based on the coupled λ exonuclease cleavage reaction as well as the FRET between dye labeled DNA and GO. Sensitive detection of PNK activity and inhibitor screening were achieved due to the extraordinary fluorescence quenching ability of GO and the efficient cleavage of double-stranded DNA (dsDNA) with 50 -phosphoryl termini by λ exo which is a highly processive 50 -30 enzyme catalyzing the removal of 50 mononucleotides from duplex DNA to generate single-stranded DNA (ssDNA) and mononucleotides. Because of the different interaction intensity of ssDNA, dsDNA with GO, dye-labeled dsDNA exhibits strong fluorescence emission, while quite low fluorescence signal is observed when the phosphorylated double helixes are cleaved by λ exo, providing a high signal-to-background ratio. In addition, unlike the complexity of the hairpin DNA probe design, the design of the DNA probe in our experiments is not strictly limited. Compared to the traditional methods, the asproposed strategy is convenient but with high analytical performance. The method is not only meaningful for further research on the disease-related biochemical process but also valuable to the molecular-target therapies and the nucleotide kinase-target drug discovery.

’ EXPERIMENTAL SECTION Reagents. Graphite (99.9%, 325 mesh) was purchased from Alfa Aesar. T4 polynucleotide kinase (10 units/μL), λ exonuclease (5 units/μL) were obtained from New England Biolabs (NEB, U.K.). Tris(hydroxymethyl)aminomethane (Tris), adenosine triphosphate (ATP), dithiothreitol (DTT), and adenosine diphosphate (ADP) were bought from Beijing DingGuo Biotech. Co., Ltd. The DNA sequences were purchased from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. Sequences of oligonucleotide probes used in this work are listed as follows: Probe 1 (P1-FAM), 50 -FAM-GTA CAC AGA CTC AGC-30 ; Probe 2 (P2), 50 -GCT GAG TCT GTG TAC-30 . Synthesis of GO. GO was synthesized according to previous reports.29,30 Generally, the preoxidized graphite was mixed with 12 mL of concentrated H2SO4 at 0 °C. Then 1.5 g of KMnO4 was added slowly under stirring in an ice-bath. After stirring at 35 °C

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for 4 h, 100 mL of deionized water was added to dilute the mixture. A volume of 2 mL of 30% H2O2 was then added drop by drop. Next, synthesized GO was filtered and washed with 0.1 M HCl(aq) and deionized water at least 5 times. The obtained solid was redispersed into deionized water followed by dialysis for 7 days. The resulting solution was filtrated and finally dried in vacuum to obtain GO powder. GO powder (10 mg) was dispersed in 5 mL of deionized water by sonication for 2 h. The mixture was then centrifuged at 5000 rpm for 20 min and the supernatant was diluted further, yielding a stable dark yellow GO dispersion with the concentration about 0.1 mg/mL. DNA Hybridization and Fluorescence Quenching Assay. The hybridization between P1-FAM (1 μM) and P2 (1 μM) was carried out in Tris-HCl buffer (20 mM Tris, 300 mM NaCl, pH 8.0) for 1 h (48 °C). The obtained dsDNA-FAM solution (about 1 μM) was stored at 4 °C for further use. To evaluate the fluorescence quenching of P1-FAM and dsDNA-FAM on the GO surface, 80 nM P1-FAM, and 80 nM dsDNA-FAM were prepared in the reaction buffer (70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT, pH 8.0) containing 10 units of λ exo, respectively. After the addition of a different amount of GO (0.1 mg/mL), the fluorescence of the mixture was measured at 520 nm with the excitation of 497 nm at room temperature. PNK-Catalyzed Phosphorylation and Assay Optimization. In a typical phosphorylation and cleavage assay, 80 nM dsDNAFAM, 0.1 mM ATP, 10 units of λ exo, and a certain amount of PNK were put into 100 μL of reaction buffer. After the incubation at 37 °C for 30 min, 10 μL GO (0.1 mg/mL) was added into the reaction mixture, followed by the fluorescence measurement with the excitation wavelength of 497 nm. The concentration optimizations of λ exo, ATP, and Mg2+ were 0.00110 units, 0.0015 mM, and 0.130 mM, respectively. The reaction pH was optimized from 7.2 to 9.6. Kinase Activity Detection in Buffer and Cell Extracts. In a typical kinase activity detection using cell extracts, 1% (v/v) cell extracts was added in the reaction buffer with all the other conditions the same as the description mentioned above. Whole cell extracts were prepared from HeLa cells according to the previous reports,31,32 which were obtained from a human epithelial carcinoma cell line. HeLa cells were cultured in DMEM/High Glucose medium (HyClone, Thermo Scientific) supplemented with 10% fetal calf serum (Zhejiang Tianhang Biological Technology Co., Ltd.) in an incubator (5% CO2, 37 °C). Kinase Inhibitor Evaluation. In the inhibition experiment, to evaluate the effects of inhibitors on the PNK-catalyzed phosphorylation process, several kinds of inhibitors, including adenosine diphosphate (0.110 mM), (NH4)2SO4 (130 mM), and Na2HPO4 (550 mM), were also contained in the reaction buffer, respectively. After the addition of 80 nM dsDNA, 0.1 mM ATP, 10 units of λ exo, and 1 unit of PNK, the reaction was performed at 37 °C for 30 min. The following procedures were similar as above. Instruments. The fluorescence spectra were obtained via a Hitachi F-7000 fluorescence spectrophotometer. Transmission electron microscopy (TEM) images were recorded using a Hitachi H-7650 TEM opened at an accelerating voltage of 80 kV. XRD patterns were taken out via a D8 Advance (Bruker) X-ray diffractometer using Cu Kα radiation (λ = 1.5418 Å). Raman spectra were collected on a RM 2000 microscopic confocal Raman spectrometer (Renishaw PLC, England) with a 633 nm HeNe laser beam. Circular dichroism (CD) measurements were taken with a JASCO J-815 spectrometer (JASCO International Co. Ltd., Tokyo, Japan) 8397

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Scheme 1. Schematic Representation of GO Based Platform for PNK Activity and Inhibition Analysisa

a

(A) Hybridized dsDNA-FAM was phosphorylated by PNK and then immediately cleaved by λ exo. Fluorescence of the resulting ssDNAFAM was greatly quenched by the addition of GO. (B) When dsDNAFAM was only mixed with λ exo under the same reaction conditions, it maintained its original structure and most of the fluorescence intensity in the presence of GO.

at room temperature. Fluorescence polarization were measured on a multilabel plate reader (Perkin Elmer) at room temperature.

’ RESULTS AND DISCUSSION Strategy for PNK Activity Detection. The developed strategy for investigating PNK activity is demonstrated in Scheme 1. FAM attached dsDNA (dsDNA-FAM), which acts as a sensitive signal report probe, was obtained by hybridization between the FAM labeled oligonucleotide probe 1 (P1-FAM) and oligonucleotide probe 2 (P2). Most of the fluorescence of dsDNA-FAM catalyzed only by λ exo still remained after mixing with a certain amount of GO. The reason for the fact is that λ exo exhibits a slow cleavage rate to dsDNA with the 50 -hydroxyl end and the adsorption between dsDNA-FAM and GO is weak and unstable. Nevertheless, when the dsDNA-FAM was phosphorylated by PNK at the 50 -hydroxyl end, the resulting 50 -phosphoryl termini product was then promptly cleaved by λ exo, yielding a FAM labeled ssDNA (i.e., P1-FAM). The fluorescence was greatly quenched after the addition of GO originated from the strong adsorption of ssDNA on GO and the effective FRET between dye and GO. Thus the activity of PNK can be easily reflected by the fluorescence signals change. Interactions Between FAM-Attached DNA and GO. Fluorescence emission spectra are used to investigate the fluorescence quenching of dye resulting from the interaction between FAMlabeled DNA and GO. It is observed from Figure 1A, P1-FAM exhibited strong fluorescence emission around 520 nm in the absence of GO. However, the fluorescence intensity gradually decreased with the increase of GO concentration. Up to 95% fluorescence emission was quenched upon the addition of 10 μL of GO within 5 min (Figure 1C). The fact is attributed to the strong adsorption of the ssDNA on the GO surface and the super fluorescence quenching ability of GO originated from the effective FRET between dye and GO.33 While, as a comparison, 80% fluorescence still remained when dsDNA-FAM was mixed with GO under the same experimental conditions (Figure 1B). It is mainly due to the weak and unstable adsorption of dsDNAFAM on the GO surface, which inhibited the FRET between the

Figure 1. (A) Fluorescence spectra of 80 nM P1-FAM after incubation with different amounts of 0.1 mg/mL GO (top to bottom: 0, 3, 6, 9, 10 μL) for 5 min. (B) Fluorescence spectra of 80 nM dsDNA-FAM after incubation with 0.1 mg/mL GO (top to bottom: 0, 10 μL) for 5 min. (C) Fluorescence intensities of (a) 80 nM P1-FAM and (b) 80 nM dsDNA-FAM via time in the presence of 10 μL of GO. The assays were all carried out in the reaction buffer containing 10 units of λ exo.

dye and GO.20 It was noted that 3 μL of GO was enough for quenching equivalent P1-FAM in the reaction buffer without 10 units of λ exo (Figure S2 in the Supporting Information). The reduction of GO usage may be attributed to the adsorption between enzyme and GO through noncovalent interactions such 8398

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Figure 2. Fluorescence spectra of 80 nM dsDNA-FAM with (a) and without (b) phosphorylation after addition of 10 μL of GO in the reaction buffer. The concentrations of ATP, λ exo, and PNK were 0.1 mM, 10 units, and 10 U mL1, respectively.

as hydrogen bonding,34,35 which blocks the interaction sites on GO surface providing for ssDNA. In order to further investigate the interaction between ssDNA, dsDNA, and GO, fluorescence polarization (FP) and circular dichroism (CD) were used (Figure S3 in the Supporting Information). FP measurements provide information on molecular orientation, mobility, and interaction processes based on the change in molecular weight of the fluorophore. Specifically, when the fluorophore binds with other materials, FP values will increase as its molecular weight increases. Consequently, FP has been frequently utilized to investigate the binding ability of ssDNA and dsDNA on the GO surface.36 Figure S3A in the Supporting Information reveals that the FP of ssDNA-FAM was significantly increased when GO was introduced, while little difference was found in that of dsDNA-FAM. The data indicate a stronger binding affinity of ssDNA-FAM onto the GO surface, which is consistent with the results obtained from the fluorescence method. CD spectra provide more evidence for the interaction between DNA and GO. CD spectroscopy measures the differential absorption of left-handed polarized light and right-handed polarized light which arises from structural asymmetry. An ordered structure results in a CD spectrum which contains both negative and positive signals. As known, ssDNA generally exhibits characteristic negative and positive peaks at 276 and 249 nm, respectively.37 It is observed from Figure S3B in the Supporting Information that after incubation with GO (>12 h), both the intensity of the negative and positive peaks decreased, suggesting the conformational transformation of ssDNA upon adsorption to GO. On the contrary, the typical bands of dsDNA undergo little change with the addition of GO (Figure S3C in the Supporting Information), which indicates a weak interaction between dsDNA and GO. In combination with fluorescence and FP data, all the results suggest that the binding affinity of ssDNA and GO is much stronger than that of dsDNA and GO, which is the basis for the following PNK activity analysis. Monitoring of the PNK-Catalyzed Phosphorylation. Figure 2 manifests the fluorescence emission of dsDNA-FAM after the coupled enzyme reaction with (curve a) and without (curve b) PNK using GO as a sensing platform. As it is shown, dsDNAFAM catalyzed only by λ exo exhibits high fluorescence signal in

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the presence of 10 μL of GO. However, when dsDNA-FAM was reacted with both PNK and λ exo, a much lower fluorescence was observed. The fluorescence intensity of dsDNA-FAM catalyzed by PNK and λ exo was 10 times smaller than that of dsDNAFAM cleaved by λ exo only. The main reason for this fact is that PNK catalyzes the γ-phosphate residue of ATP transferring to the 5 0 -hydroxyl group of dsDNA-FAM, yielding a phosphate moiety at the 5 0 -dsDNA-FAM end. Also then λ exo rapidly degrades dsDNA-FAM with 5 0 -phosphoral at a speed of 12 nucleotides/s approximately,38,39 which is over 300 times more quickly than that of dsDNA with the 50 -hydroxyl end40 owing to the formation of an inert substrateenzyme complex.38 As a result, under the same reaction condition, dsDNA-FAM phosphorylated by PNK was rapidly hydrolyzed by λ exo, leaving dye labeled ssDNA (P1-FAM), while dsDNA-FAM without 50 -phosphoral still remained unchanged. The different adsorption abilities of P1-FAM and dsDNA-FAM on the GO surface lead to the significant discrepancy of the fluorescence signals. The results suggest that DNA phosphorylation is the rate determining step in the coupled enzyme reaction system, and the accurate and rapid kinase activity measurement can be achieved through the PNK-λ exo system and the GO based platform. Optimization of Assay Conditions. The reaction time is a crucial parameter for the PNK-catalyzed phosphorylation and the coupled λ exo cleavage process. An excess of incubation time would make the fluorescence signals unreliable as λ exo would also hydrolyze dsDNA with 50 -hydroxyl slowly.40 Figure 3A displays the changes of fluorescence intensity with incubation time. It was observed that the fluorescence signal decreased gradually with the increase of the reaction time and then reached equilibrium in 30 min, suggesting the complete phosphorylation and cleavage process. In addition, as can be seen from Figure 3B, over 90% fluorescence emission was quenched when 10 units of λ exo was used. Thus, the optimal reaction time and the amount of λ exo were chosen to be 30 min and 10 units, respectively. In addition, the PNK and λ exo coupled reaction is also sensitive to pH and the concentration of Mg2+. In our system, the optimum reaction buffers for these two enzymes are quite different. As a result, both pH and Mg2+ concentration are optimized in our expriments. As shown in Figure S4 in the Supporting Information, the optimal pH and the concentration of Mg2+ for the coupled enzyme catalyzed procedure were 8.0 and 10 mM in these experiments, respectively. It is known that the phosphate group at the 50 -DNA end is offered by ATP during the phosphorylation process, and the absence of ATP will result in the blockage of phosphorylation.1 The effect of ATP concentration on kinase activity is shown in Figure 3C. As can be seen, the fluorescence intensity decreased with the increasing concentrations of ATP and then reached its minimum at the ATP concentration of 0.1 mM. When the concentration of ATP was higher than 0.1 mM, the fluorescence signal increased again. This inhibition effect is a consequence of the competitive binding between ATP and DNA to PNK, whose binding sites for DNA are partially blocked by ATP. Thus, the optimal concentration of ATP was chosen to be 0.1 mM. Fluorescence Measurement of PNK Activity. For the sake of evaluating the activity of PNK, different concentrations of PNK were applied in the PNK-λ exo coupled enzyme reaction based on the optimal assay conditions. Figure 4A shows the fluorescence responses for different PNK concentrations. It is observed that the fluorescence signals gradually decreased as the concentrations of PNK varied from 0.05 to 10 U mL1. The amount of 8399

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Figure 4. (A) Fluorescence intensitywavelength curves with different activity units of PNK (top to bottom, 0, 0.05, 0.1, 0.5, 1, 5, 10 U mL1) in reaction buffer (inset, dependence of fluorescence intensity on the logarithm of PNK concentration). (B) The fluorescence intensity with different activity units of PNK in reaction buffer containing 1% (v/v) cell extracts. The concentrations of dsDNA-FAM, ATP, and λ exo were 80 nM, 0.1 mM, and 10 units, respectively.

Figure 3. (A) Optimization of the reaction time. The concentrations of ATP and λ exo were 0.1 mM and 10 units, respectively. (B) Optimization of λ exo concentration. The concentration of ATP was 0.1 mM. (C) Optimization of ATP concentration. The concentration of λ exo was 10 units. The assays were all carried out in the reaction buffer, containing 80 nM dsDNA-FAM and 10 U mL1 PNK (S/N = 3).

PNK was reflected by the change of fluorescence signal, which originated from the FRET between dye attached DNA and GO. The dependence of fluorescence signal on PNK concentration is displayed in Figure 4B. The fluorescence signal was linearly decreased with the logarithm of PNK concentration in the range from 0.05 to 10 mL1. The detection limit of PNK was 0.05 mL1 (signal-to-noise ratio of 3), which was lower than that of the radical isotope 32P-labeling methods and comparable to the results obtained from previous fluorescence assays (Table S1 in the

Supporting Information). The linear relationship can be described as F = 197.1149.9 log c with the correlation coefficient of R2 = 0.985, where F is the fluorescence intensity and log c is the logarithm of the PNK concentration. This demonstrates that the coupled enzyme reaction system and the GO based platform can be applied to sensitive kinase activity analysis in a wide concentration range. Intracellular phosphorylation of 50 -hydroxyl DNA is an essential step in nucleic acid repair and replication. In order to examine the possibility of the as-proposed sensing platform for cellular PNK activity profiling, HeLa cell extracts were added in the buffer to simulate the intracellular environment during the test procedure. Figure S5 in the Supporting Information demonstrates that in the cell extracts-containing buffer, 10 μL of GO was enough to quench 90% fluorescence emission of dye-labeled ssDNA, while double helixes still preserved 80% fluorescence intensity under the same conditions. These phenomena are in good agreement with those in the reaction buffer without cell extracts. It is observed from Figure 4B that the fluorescence signals decreased when the concentrations of PNK gradually increased from 0.1 to 10 U mL1. The fluorescence intensity and the logarithm of PNK concentration also exhibits a linear relationship like that operated in PBS solution (Figure S6 in the Supporting Information). The above results demonstrate that 8400

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out with inhibitor at different concentrations in the reaction buffer. Figure 5A shows that fluorescence intensity increased with the increasing concentrations of ADP, indicating the weakening of DNA phosphorylation. Merely 0.1 mM ADP had led to an obvious inhibition effect on PNK. On the basis of the results, the addition of 1.0 mM ADP had caused a 50% decrease in DNA phosphorylation. This is owing to the reversible phosphorylation reaction when ADP and 50 -phosphoryl nucleic acids exist in the reaction buffer simultaneously.1 In addition, as shown in Figure 5B,C, ammonium sulfate and sodium hydrogen phosphate were further used to evaluate the salt effects on PNK. As expected, increasing concentrations of ammonium sulfate or sodium hydrogen phosphate both resulted in the decrease of fluorescence intensity. The amount of ammonium sulfate and sodium hydrogen phosphate causing a 50% fluorescence decrease was 10 and 20 mM, respectively. The effect of the three small molecules on PNK activity is similar to that reported in previous literature.1,14 Here, there are two possible reasons to explain the salt effect on kinase activity.41,42 First, at high salt concentrations, the structure of dsDNA is known to be more stable, which probably inhibits the activity of the 50 -hydroxyl group. Second, the enzyme conformation may be significantly affected by a high concentration of salts. The conformation change of the enzyme may further reduce the activity of PNK as well as the affinity between kinase and its substrates. These results indicate that the proposed strategy is promising in quantitatively monitoring the activity of the kinase inhibitors.

’ CONCLUSIONS In summary, the activity and inhibition of PNK were sensitively and rapidly detected using the coupled λ exonuclease cleavage reaction and the GO-based sensing platform. The efficient cleavage capacity of λ exo and the super quenching ability of GO both contribute to the sensitive screening of PNK activity. The as-proposed method provides a wide linear range from 0.05 to 10 U mL1 and a low detection limit of 0.05 U mL1 for PNK activity analysis. In addition, this strategy is cost-effective and avoids the complex design of the hairpin DNA probe. Furthermore, the inhibition effects of ADP, ammonium sulfate, and sodium hydrogen phosphate on phosphorylation can be evaluated accurately and conveniently. Given the crucial roles of kinases in some biological processes, the sensitive and universal GO based sensing platform is promising in developing on-chip, high-throughput assays for drug discovery and clinical diagnostics. Figure 5. Inhibition effects of (A) ADP, (B) (NH4)2SO4, and (C) Na2HPO4 on phosphorylation. The assays were carried out in the reaction buffer, containing 80 nM dsDNA-FAM, 10 U mL1 PNK, 0.1 mM ATP, and 10 units λ exo.

’ ASSOCIATED CONTENT

the as-proposed sensing platform works well in the intracellularmimicking environment, suggesting that the method could be further used for real sample analysis. PNK Activity Inhibition Evaluation. The inhibition effects of adenosine diphosphate (ADP), ammonium sulfate, and sodium hydrogen phosphate on phosphorylation have been investigated through the proposed detection platform. These chemicals are considered to have no inhibition effect on the activity of λ exo and,17 thus, can be used as inhibitors for PNK. To evaluate the inhibition effects on PNK activity, each inhibition assay was carried

’ AUTHOR INFORMATION

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Corresponding Author

*Phone: 86-10-62795290. Fax: 86-10-62771149. E-mail: jhli@ mail.tsinghua.edu.cn.

’ ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China (Grant No. 21005046), the National 8401

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Analytical Chemistry Basic Research Program of China (Grant No. 2011CB935700), and the Tsinghua University Initiative Scientific Research Program.

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