Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based

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Sensitive detection of polynucleotide kinase activity by paperbased fluorescence assay with # exonuclease-assistance Hua Zhang, Zhen Zhao, Zhen Lei, and Zhenxin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03567 • Publication Date (Web): 31 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Analytical Chemistry

Sensitive Detection of Polynucleotide Kinase Activity by Paper-Based Fluorescence Assay with λ ExonucleaseAssistance Hua Zhang†, Zhen Zhao†,‡, Zhen Lei†,‡, Zhenxin Wang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China. ‡ University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, P. R. China *Tel.: +86 431 85262243. Fax: +86 431 85262243. E-mail: [email protected] ABSTRACT: The phosphorylation of nucleic acid with 5ʹ-OH termini catalyzed by polynucleotide kinase (PNK) invovles several significant cellular events. Here a paper-based fluorescence assay with λ exonuclease-assistance was reported for facile detection of PNK activity through monitoring the change of fluorescence intensity on paper surface. Cy5-labeled ssDNA was firstly immobilized on the surface of aldehyde group modified paper. And BHQ-labeled ssDNA was then employed to quench the fluorescence of the immobilized Cy5-labeled ssDNA with the help of an adaptor ssDNA. When PNK and λ exonuclease cleavage reaction were introduced, the fluorescence quenching effect on the paper surface was blocked because of the digestion of phosphorylated dsDNA by the coupled enzymes. By using this paper-based assay, PNK activity both in pure reaction buffer and in practical biosample have been successfully measured. Highly sensitive detection of PNK activity down to 0.0001 U mL-1 and lysate of ca. 50 cells is achieved. The inhibition of PNK activity has also been investigated and a satisfactory result is obtained.

formats for paper-based assay have been developed, such as microfluidic PAD, lateral flow device and dipstick assays.27-30 Importantly, the three-dimensional microstructures of paper enable the liquid transport easily via capillary wicking without the requirement of an external power. This movement of sample through pores of micrometer dimensions is beneficial for saving assay time.31 Moreover, the paper surface can be modified with varieties of functional groups, which can be further used to immobilize various small molecules or biological macromolecules with very small sample volumes.32 Therefore, PAD is regarded as a low-cost and effective alternative substrate and holds great potential for POC diagnosis.33-35 Herein, we designed a paper-based assay for the detection of PNK activity and inhibition coupled with λ exonuclease cleavage reaction on the basis of fluorescence quenching. The λ exonuclease, as a highly processive 5ʹ-3ʹ dsDNA exonuclease, tends to digest dsDNA with a phosphate moiety at 5ʹ-ends and shows very low activity on non-phosphorylated dsDNA. A sandwich DNA hybridization system was constructed to achieve fluorescence quenching. The fluorescence intensity on paper surface could be easily obtained by a fluorescence microarray scanner. As shown in Figure 1, Cy5-labeled DNA (P1-Cy5) was first immobilized on the surface of aldehyde group modified paper through covalent attachment. And BHQlabeled DNA (P2-BHQ) was added onto the paper to quench the fluorescence with the help of an adaptor DNA (P3). In the absence of PNK, the phenomenon of fluorescence quenching is negligibly affected by λ exonuclease cleavage since λ exonuclease shows low catalytic efficiency towards ssDNA or dsDNA with 5ʹ-hydroxyl end, and it can’t begin to digest DNA from the nick or gap of dsDNA. When both of PNK phosphorylation and λ exonuclease cleavage reaction were

INTRODUCTION T4 polynucleotide kinase (PNK), first discovered in 1965,1 is a primary member of 5ʹ-kinase family that can catalyze the transfer of γ-phosphate residue of ATP to the 5ʹ-hydroxyl group of oligonucleotides and nucleic acids.2,3 The phosphorylation process involves several significant cellular events, such as nucleic acid metabolism, DNA replication, DNA recombination and DNA repair during strand damage and interruption.4-6 The hindrance of DNA phosphorylation will result in serious diseases including loom’s syndrome, Werner syndrome, and Rothmund-Thomson syndrome.7,8 In addition, PNK might be a promising target in the radio therapy of cancer, since PNK inhibitors are able to increase the sensitivity of human tumors to γ–radiation.9,10 Therefore, it is of great importance to develop PNK activity detection assays for biological and biomedical research. The traditional PNK activity assays including radioactive isotope 32P-labeling, polyacrylamide gel electrophoresis (PAGE) and autoradiography11,12 have several limitations such as time-consuming, high cost, radioactive contamination, and requirement of skillful technicians. To overcome these disadvantages, varieties of other techniques for the determination of PNK have been developed including colorimetric,13,14 fluorescent,15-20 electrochemical,21,22 chemiluminescent,23 and so on. Nevertheless, the above techniques are mainly based on solution-phase reaction systems and need complicated instruments, which are not practical for using outside laboratory and can’t meet the demands of point-of-care (POC) test. Paper-based analytical device (PAD) has attracted significant attention in recent years due to its low-cost, portability, flexibility, disposability, and biocompatibility.24-26 Many kinds of

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introduced, the 5ʹ-hydroxyl end of P2-BHQ was firstly phosphorylated by PNK, and then the phosphorylated product (phosphorylated BHQ-adaptor dsDNA) was rapidly cleaved by λ exonuclease. The sandwich hybridization system was broken by the enzymatic reactions, resulting in a low quenching efficiency. As a consequence, the PNK activity can be easily and sensitively reflected by the change of fluorescence intensity on the paper surface. Comparison with literature reported solution phase systems, the paper-based fluorescence assay gives more conductive to store the experimental results.

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read by LuxScan-10K fluorescence microarray scanner (CapitalBio Ltd., Beijing, China). Fluorescence quenching assay. To block the non-reacted aldehyde groups, the paper substrate was immersed in methoxypolyethylene glycol amine (PEG-NH2, 1.0 mg mL-1 in Milli-Q water) solution and incubated for 1 h, washed with 30 mL Milli-Q water (3 times) and dried in air. Then, 1.5 µL aliquot of quenching solution (consisted of P2-BHQ and P3) was transferred to the P1-Cy5 modified test zone and incubated at 37 °C under 60% humidity for 30 min. Subsequently, the paper device was washed and dried as previously described. In order to obtain the optimal quenching condition, different molar ratios (1:3, 1:2, 1:1, 2:1, 3:1) of P2-BHQ and P3 were used. PNK activity detection in buffer. In a typical phosphorylation and cleavage assay, 2 µM P2-BHQ, 1 µM P3, 1 mM ATP, 1 U µL-1 λ exo, and a certain amount of PNK were added into 10 µL of reaction buffer and incubated at 37 °C for 30 min. Then the reaction mixture was heated to 90 °C for 5 min to deactivate the enzymes and gradually cooled to 37 °C. Subsequently, 1.5 µL aliquot of the mixture was transferred onto test zone and incubated at 37 °C under 60% humidity for another 30 min. Finally, the paper substrate was washed with test buffer and water, followed by scanning with fluorescence microarray scanner. PNK activity detection in cell lysates. HeLa (human cervical cancer cell), A549 (human lung adenocarcinoma cell), HL7702 (human normal liver cell) cells were cultured with fresh Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum in a humidified 5% CO2 incubator at 37 °C. The cell extracts were prepared according to the previous reports37. Generally, cells in exponential growth were washed with phosphate buffer saline (PBS) three times and resuspended at 106 cells/10 µL in buffer A (10 mM Tris-HCl, pH=7.8, 200 mM KCl). An equal volume of lysis buffer B (10 mM Tris-HCl, pH=7.8, 600 mM KCl, 2 mM EDTA, 2 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulphonyl fluoride (PMSF),40% (v/v) glycerol, 0.2% (v/v) Nonidet P-40) was added to the cell suspension. After incubated at 4 °C for 1.5 h with occasional shake to allow cell lysis, the mixture was spun at 16000 g for 10 min to remove the cell debris, and the supernatant were collected. Different dilution ratio of the supernatant was added to the PNK detection assay solution with the same procedure in pure reaction buffer. Kinase inhibition assay. ADP and (NH4)2SO4 were used as the model inhibitors of PNK to evaluate the inhibition effects. In a typical inhibition assay, different concentrations of inhibitors were added into the reaction buffer respectively. After addition of 2 µM P2-BHQ, 1 µM P3, 1 mM ATP, 5 U mL-1 PNK and 1 U µL-1 λ exo, the procedures were as similar as mentioned above. Kinase selectivity assay. In order to investigate the specificity of the proposed method for detection of PNK, EcoRI / BamHI / EcoRV / HpaII / heat-inactivated PNK were respectively added into the reaction buffer instead of PNK. The mixture of all kinds of enzymes was also tested. The following procedures were similar to that stated above.

Figure 1. The illustration of the designation and principle of the paper-based fluorescence assay for PNK activity analysis.

EXPERIMENTAL SECTION Reagents. Whatman cellulose chromatography paper (Grade 1) with a 0.18 mm thickness was purchased from Sigma-Aldrich Company (St. Louis, USA). T4 polynucleotide kinase (10 units/µL), λ exonuclease (5 units/µL), adenosine triphosphate (ATP) were purchased from New England Biolabs Ltd. (Hitchin, U.K.). Tris(hydroxymethyl)aminomethane (Tris), dithiothreitol (DTT) were purchased from Beijing DingGuo Biotech. Co., Ltd. (Beijing, China). The oligonucleotides (P1-Cy5: 5′-Cy5-TGTTGTTGGG-T10-NH2-3′; P2BHQ: 5′-CGAGGCTGCACT-BHQ2-3′; and P3: 5′AACCCAACAACATAGTGCAGCCTCG-3′) and adenosine diphosphate (ADP) were obtained from Shanghai Sangon Biological Engineering Technology&Services Ltd. (Shanghai, China). Sodium periodate (NaIO4), lithium chloride (LiCl), ammonium sulfate ((NH4)2SO4) and other reagents were of analytical grade, and purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). All solutions were prepared with Milli-Q water (18.2 MΩ cm). Preparation of test zones and immobilization of P1-Cy5. The paper was firstly treated by NaIO4 for forming aldehyde groups36, which is used to immobilize the P1-Cy5 through Schiff alkali reaction. Briefly, 0.5 g Whatman cellulose chromatography paper was immersed into 100 mL solution containing 50 mM NaIO4 and 700 mM LiCl, and incubated at 55 °C in dark chamber for 3 h, washed with 50 mL Milli-Q water (three times) and dried in oven at 40 °C. Then, the dried paper was cut into pieces, and patterned according to a template to make circular test zone. Subsequently, 1.5 µL aliquot of 200 nM P1-Cy5 were transferred to the test zone and incubated at 37 °C under 60% humidity for 1 h, washed with 30 mL 50 mM Tris-HCl buffer (pH=8.0, twice) and 30 mL Milli-Q water (twice), respectively. After dried in air, the paper substrate was adhered to a piece of glass slide using double-sided tape, and

RESULTS AND DISCUSSION The design of paper device. The paper was first functionalized with aldehyde groups by periodate (IO4-) oxidation of cellulose for the immobilization of amine-functionalized P1Cy5. The SEM images (Figure S1) show that the cellulose

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Analytical Chemistry hydroxyl DNA. However, DNA phosphorylation could be inhibited by high concentration of ATP since the binding site of PNK is partially blocked by the excess ATP.39 The influence of ATP concentration on PNK activity is illustrated in Figure 2C. The minimum value of fluorescence quenching efficiency is obtained at 1 mM ATP in the reaction mixture. In addition, the fluorescence quenching efficiency on the test zone is decreased with increasing the concentration of λ exonuclease, and reached saturation at 1 U µL-1 λ exonuclease in the reaction mixture (Figure 2D). Detection of PNK activity. On the basis of the optimal experimental conditions (the molar ratio of P2-BHQ and P3 at 2:1, 30 min phosphorylation reaction time, 1 mM ATP and 1 U µL-1 λ exonuclease), various concentrations of PNK were applied to evaluate the sensitivity of proposed paper-based fluorescence assay. As shown in Figure 3A (black line), the

paper consists of fabric ribbons and there is no obvious structural difference between the original and treated paper. After periodate oxidation, an absorption band at 1726 cm-1 derived from C=O stretching can be observed in the FT-IR spectrum (Figure S2).36 Furthermore, a new peak at 288.4 eV is observed in the C 1s XPS spectrum of treated paper, which is assigned to C=O bond (Figure S3).38 These results suggest the successful functionalization of paper with aldehyde groups. The IO4- treated paper was cut into pieces with dimensions of 20 mm × 60 mm (width × length) and patterned according to a template (Figure S4). Permanent marker was used to make circular test zones (ca. 2.5 mm in diameter) forming a hydrophobic barrier. Under this format, each assay just requires 1.5 µL sample solution. The fluorescence images of test zones can be easily read by a fluorescence microarray scanner, and the fluorescence intensity was calculated using Image J software (National Institute of Health, USA). In this case, the fluorescence intensity of test zone with P1-Cy5 is defined as F0, while the fluorescence intensity of test zone quenched by P2BHQ mixture is defined as F. Thus the fluorescence quenching efficiency can be obtained through (F0-F)/F0.

Figure 2. Optimization of experimental conditions. The effects of (A)the molar ratio of BHQ-DNA and Adaptor-DNA, (B) the enzyme reaction time,(C) ATP concentration, and (D) λ exonuclease concentration on the quenching efficiency. The assays were all carried out in the reaction buffer containing 5 U mL-1 PNK. Error bars are standard deviations (S/N=3).

Optimization of assay conditions. Series of experimental conditions including the ratio of P2-BHQ and P3, phosphorylation time, adenosine triphosphate (ATP) concentration and λ exonuclease concentration were optimized for generating reasonable assay performance. Firstly, after immobilization of P1-Cy5 on the paper, the mixtures of P2-BHQ and P3 with different molar ratio were applied to different test zones, respectively. As shown in Figure 2A, the highest quenching efficiency is achieved with the molar ratio of P2-BHQ and P3 at 2:1. As expect, λ exonuclease only shows negligible effect on the fluorescence quenching efficiency because λ exonuclease can not cleave the non-phosphorylated DNA (Figure S5). The phosphorylation time is a critical factor for the PNKcatalyzed phosphorylation and the coupled λ exonuclease cleavage reaction process. As shown in Figure 2B, the fluorescence quenching efficiency of test zone is gradually decreased with increasing phosphorylation time, and reaches equilibrium after 30 min reaction. ATP, as the enzymatic co-substrate for PNK, plays a crucial role in the DNA phosphorylation. During the phosphorylation, ATP provides the phosphate group for 5ʹ-

Figure 3. (A) The fluorescence quenching efficiency as a function of PNK concentrations in pure reaction buffer (black line) and spiked into 50% (v/v) HeLa lysate (red line). Inset: corresponding fluorescence images of the test zones in pure reaction buffer (left) and in 50% (v/v) HeLa lysate (right). (B) The fluorescence quenching efficiency as a function of cell lysate amount. (C) Corresponding fluorescence images of the test zones after incubation with various amounts of HeLa (up), A549 (middle), and HL7702 (bottom) cell lysates. Error bars are standard deviations (S/N=3).

fluorescence intensity of test zone is gradually increased with increasing the concentration of PNK, i.e., higher concentration of PNK brought more 5ʹ-phosphorylated BHQ-Adaptor dsDNA product to be cleaved by λ exonuclease, which results in lower fluorescence quenching efficiency. The fluorescence

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quenching efficiency is found to vary linearly with the logarithm of PNK concentration in the range from 0.0001 to 5 U mL-1 (correlation coefficient R2=0.993). The detection limit of PNK is 0.0001 U mL-1 according to the 3σ rule, which is lower than or comparative with those of previous reports (Table S1).13-20,39,40 The assay-assay precision coefficient of variation (CV) of the PNK activity detection is below 5.1% in all experimental data sets, indicating that the assay has reasonable reproducibility. In addition, the detection principle is also demonstrated by gel electrophoresis analysis (Figure S6). The sandwich hybridization system was efficiently digested by the enzymatic reactions when both of PNK and λ exonuclease were introduced into the reaction mixture. The results demonstrated that the paper-based strategy can be applied to sensitively detect the PNK activity in a relatively wide concentration range. Investigation of PNK activity in cell lysate. The practicability of proposed assay was also studied by the detection of PNK activity in complex biological matrix (i.e., cell lysate). For instance, different cell lysates (HeLa, A549 and HL-7702) were added into the reaction buffer to simulate the intracellular environment during the experimental procedure (Figure S7), respectively. As shown in Figure 3A (red line), the paperbased fluorescence assay works well in the diluted cell lysate. The fluorescence quenching efficiency is linearly changed with the logarithm of spiked PNK concentration in the range from 0.0001 to 5 U mL-1 (correlation coefficient R2=0.991), which is in good agreement with that in pure reaction buffer. The results demonstrated that this assay can be applied to detect PNK in complex biosamples. In order to further test the feasibility of paper-based fluorescence assay for detecting the cellular PNK activity, expression levels of PNK activity in different cell lines were measured. In this case, three cell lines (HeLa, A549 and HL-7702) were arbitrarily selected. As expected, the fluorescence quenching efficiency is decreased with increasing the amount of cell lysate in the reaction buffer (Figure 3B). Generally, the changes of fluorescence quenching efficiencies of three cell lysates follow the order of A549＞ HeLa＞HL-7702, indicating that the cell lines express different levels of PNK. Importantly, the PNK activity could be detected as low as lysates of 10 HeLa cells, 10 A549 cells and 50 HL-7702 cells, respectively. The experimental result also suggests that the paper-based fluorescence assay has high sensitivity. It is demonstrated that the paper-based fluorescence assay shows great promise for detecting PNK activity in practical samples. Inhibition evaluation of PNK activity. It is reported that PNK inhibitors could enhance the sensitivity of human tumors to γ–radiation, which means that the inhibition of PNK activity may become a potential direction against cancers.41 To evaluate the paper-based fluorescence assay for screening the inhibitors of PNK, we investigated the effect of two model inhibitors, adenosine diphosphate (ADP) and (NH4)2SO4 on PNK activity. ADP can produce a reversible phosphorylation reaction which makes the phosphate of DNA transfer to ADP molecule.39 (NH4)2SO4 can suppress PNK activity through changing the structure of dsDNA and conformation of PNK.42 Both ADP and (NH4)2SO4 have been identified to have negligible inhibition effect on λ exonuclease activity.43 The inhibition assay was performed with various concentrations of inhibitors while the concentration of PNK was kept as constant. As shown in insets of Figure 4, the fluorescence intensity is gradually decreased with the increase of ADP and (NH4)2SO4 con-

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centrations. The result suggests that the PNK activity is strong inhibited by ADP and (NH4)2SO4. The IC50 (half-maximal inhibitory concentration) curves of ADP and (NH4)2SO4 are shown in Figure 4. The IC50 values for ADP and (NH4)2SO4 are 1.5 mM and 12.6 mM, respectively, which are comparable with the literature reported values.15-20 The result confirms that the paper-based fluorescence assay has the potential to screen PNK inhibitors as well as determine corresponding IC50 values.

Figure 4. The fluorescence quenching efficiency as a function of ADP concentrations (A) and (NH4)2SO4 concentrations (B). The insets are corresponding fluorescence images, and the concentrations of ADP and (NH4)2SO4 are increased from up to bottom, respectively. The assays were carried out in the reaction buffer containing 5 U mL-1 PNK, 1 mM ATP, and 1 U µL-1 λexo. Error bars are standard deviations (S/N=3).

Assay selectivity. The selectivity of the proposed assay is also demonstrated by detecting other enzymes including EcoRI, BamHI, EcoRV, HpaII, as well as heat-inactivated PNK under the same condition (Figure S8). Comparison with blank sample, only PNK (pure PNK or PNK in the mixture of enzyme) treated test zone gives a strong fluorescence signal and results in significant change of fluorescence quenching efficiency. The result indicates that the paper-based fluorescence assay has excellent selectivity towards PNK.

CONCLUSIONS In conclusion, we have developed a paper-based fluorescence sensing platform with λ exonuclease-assistance for highly sensitive and selective detection of PNK activity in both of pure buffer solution and native biological samples (i.e., cell lysates). This assay can also be applied to quantitative deter-

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mination of PNK inhibitor activity. This approach provides a low-cost and facile tool for studying PNK functionality and inhibition. This would open up possibilities for the future use of paper-based sensing devices in POC diagnosis and drug discovery.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Figures including (1) SEM images of the original and NaIO4 treated paper; (2) FT-IR spectra the original and NaIO4 treated paper; (3) C 1s XPS spectra of the original and NaIO4 treated paper; (4) the design of paper device; (5) the effect of λexo only on the fluorescence quenching efficiency; (6) native 12% polyacrylamide gel analysis result; (7) the standard addition method results; (8) selectivity investigation by detecting a few nonspecific enzymes with the proposed paperbased fluorescence assay. Table S1. (PDF)

AUTHOR INFORMATION Corresponding Author *Tel.: +86 431 85262243. Fax: +86 431 85262243. E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (Grant No. 21475126), Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201561) and China Postdoctoral Science Foundation (Grant No. 2016M591497).

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