A Label-Free Bioluminescent Sensor for Real-Time Monitoring

Jul 21, 2014 - Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent...
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A Label-Free Bioluminescent Sensor for Real-Time Monitoring Polynucleotide Kinase Activity Jiao Du,†,‡,§ Qinfeng Xu,‡,§ Xiaoquan Lu,*,† and Chun-yang Zhang*,‡ †

Key Laboratory of Bioelectrochemistry & Environmental analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070, China ‡ Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China S Supporting Information *

ABSTRACT: Polynucleotide kinase (PNK) plays a crucial role in maintaining the genomic stability of cells and is becoming a potential target in the radio-therapeutic treatment of cancers. The fluorescent method is usually used to measure the PNK activity, but it is impossible to obtain the real-time monitoring without the employment of the labeled DNA probes. Here, we report a label-free bioluminescent sensor for PNK activity assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. In this bioluminescent sensor, two hairpin DNA probes with 5′-protruding terminal are designed as the phosphate acceptor, and the widely used phosphate donor of ATP is substituted by dCTP. In the absence of PNK, the ligation reaction cannot be triggered due to the lack of 5′-phosphoryl groups in the probes, and the background signal is negligible. With the addition of PNK, the phosphorylation-ligation reaction of the probes is initiated with the release of AMP, and the subsequent conversion of AMP to ATP leads to the generation of distinct bioluminescence signal. The PNK activity assay can be performed in real time by continuously monitoring the bioluminescence signal. This bioluminescent sensor is much simpler, label-free, costeffective, and free from the autofluorescence interference of biological matrix, and can be further used for quantitative, kinetic, and inhibition assay. The 32P-labeling (to γ-phosphate group of ATP) is a wellestablished direct method for PNK assay.15−17 With the labels transferring from ATP donor to DNA acceptor, the increase of 32 P-labeled DNA or the decrease of ATP32 can be easily observed.18 However, this method is limited by its laborious, discontinuous monitoring and the requirement of radioactive labels. Alternatively, the nonradioactive methods such as fluorescent,19−29 colorimetric,30,31 chemiluminescent,32 and electrochemical33−36 have been developed for PNK assay. However, most of them rely on the detection of phosphorylated DNA due to the difficulty in differentiating the changes of small molecule of phosphate donor before and after the reaction. Even with the use of the labeled DNA, it is difficult to directly resolve the difference before and after DNA

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olynucleotide kinase (PNK) can catalyze the transfer of the γ-phosphate group of a nucleoside triphosphate (the most used one is ATP) to the 5′-hydroxyl termini of nucleic acids,1,2 and is a widely used reagent in nucleic acid research.3 PNK is widespread in cells and plays a critical role in the DNA repair in response to various damages, such as single-strand break,4 base excision,5 and oxidative DNA damage.6 Especially, the human PNK participates in the maintenance of DNA integrity against the endogenous and exogenous damage agents,7 and the mutations/abnormal behaviors of PNK might be closely associated with some vital human diseases.8,9 Moreover, the human PNK might become a potential target in the radiotherapeutic treatment of somatic cancers,10,11 because its small molecule inhibitors have been identified with the capability of increasing the sensitivity of human tumors to the radiotherapy.12,13 Therefore, the PNK activity assay is of great importance to the biomedical research and the drug development.14 © 2014 American Chemical Society

Received: June 18, 2014 Accepted: July 21, 2014 Published: July 21, 2014 8481

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phosphorylation without the involvement of separation.37 To enlarge such a difference, some indirect methods using PNK phosphorylation-dependent enzymatic DNA ligation19 and DNA cleavage20 have been developed for PNK assay. Because of the specific requirement of 5′-phosphorylated DNA for the activity of DNA ligase 38 and lambda exonuclease, 39 the PNK can be measured by either label19−23,27−29,33−36 or label-free24−26,30−32 detection of ligated and cleavaged DNA. The label-free method is much simpler and cheaper than the labeled method without the involvement of any complicated design, modification, and purification of DNA probes,40 but it requires multistep operation and fails to realize the real-time and rapid analysis. The fluorescence measurement is the frequently used label method for PNK assay,19−29 but it requires expensive equipment and special attention to avoid the interference of photobleaching, autofluorescence, and scattered excitation light.41 Herein, we develop a label-free bioluminescent method for PNK assay through real-time monitoring of the phosphorylation-dependent DNA ligation reaction. This method is very simple and cost-effective without the involvement of either DNA probe modification or external light excitation.

and adenylate kinase (AK) from chicken muscle were obtained from Sigma-Aldrich, Inc. (St. Louis, MO). ATP determination kit (D-luciferin, recombinant firefly luciferase, adenosine triphosphate (ATP), and dithiothreitol (DTT)) and SYBR gold were obtained from Invitrogen (Carlsbad, CA). All other reagents were of analytical grade and used as received without further purification. Corning 96-well white microplate was purchased from Fisher Scientific (Pittsburgh, PA). Ultrapure water obtained from a Millipore filtration system was used throughout all experiments. Gel Electrophoresis. All hairpin probes were prepared by heating individually to 95 °C for 5 min, followed by slowly cooling to room temperature over 40 min. In the validation experiments of phosphorylation-ligation reaction, the PNK (2.0 U) was added into the mixture of annealed probes (1.0 μM), nucleoside triphosphate (1.0 mM), E. coli ligase (1.2 U), and NAD+ (30 μM) in 10 μL of 1× Cutsmart buffer, and then incubated for 30 min at room temperature. The resultant reaction solutions (10 μL) were prestained by SYBR gold, and then loaded on a 15% native polyacrylamide gel. After electrophoresis was performed at 110 V for 70 min in 1× Tris-borate-EDTA (TBE) buffer, the image of gel electrophoresis was obtained via a Kodak 4000MM (Rochester, NY). PNK Aactivity Assay and Inhibition Assay. In a typical bioluminescence measurement, the AMP-to-ATP conversion buffer containing 1 μL of AK (1 U/μL), 1 μL of PK (1 U/μL), 1 μL of PEP (4.8 mM), and 1 μL of dCTP (39 mM), and the ATP detection buffer containing 5 mM D-luciferin, 12.5 μg/mL firefly luciferase, 250 mM Tricine buffer (pH 7.8), and 1 mM DTT were prepared prior to the phosphorylation-ligation reaction. For the real-time monitoring of PNK, the PNK was added into the mixture of annealed probes (6.0 μL, 1.0 μM), E. coli ligase (1.2 U), NAD+ (1 μL, 30 μM), the AMP-to-ATP conversion buffer (4.0 μL), and the ATP detection buffer (0.5 μL) in 60 μL of 1× Cutsmart buffer, and then the bioluminescence signal was continuously recorded with a Glomax 96 microplate luminometer (Promega, Madison, WI). For the PNK inhibition assay, both (NH4)2SO4 and Na2HPO4 were used as the model inhibitors. Various concentrations of inhibitors were separately mixed with 8 μM DNA probe and 10 U/mL PNK in 10.0 μL of 1× Cutsmart buffer. The phosphorylation reaction was performed at 37 °C for 30 min, followed by termination through heating to 95 °C for 5 min. Prior to the bioluminescence assay, the reaction solution was diluted 200 times to eliminate the detrimental effect of salt on the activity of ligase and firefly luciferase. Briefly, 0.3 μL of reaction solution was mixed with the AMP-toATP conversion buffer (4 μL), E. coli ligase (1.2 U), NAD+ (1.0 μL, 30 μM), and the ATP detection buffer (0.5 μL) in 60 μL of 1× Cutsmart buffer, and then subjected to the bioluminescence measurement. For the real sample analysis, the HEK293T cell (human embryonic kidney cells) extracts were prepared using a cell nucleoprotein extract kit (Shanghai Sangon, China) according to the manufacturer’s protocol. After an aliquot of 1.0 μL of cell extracts was added into the mixture of annealed probes (6.0 μL, 1.0 μM), E. coli ligase (1.2 U), NAD+ (1.0 μL, 30 μM), the AMP-to-ATP conversion buffer (4.0 μL), and the ATP detection buffer (0.5 μL) in 60 μL of 1× Cutsmart buffer, the bioluminescence signal was continuously recorded using the microplate luminometer (Promega, Madison, WI).

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EXPERIMENTAL SECTION Materials. The DNA sequences (Table 1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The various Table 1. Sequences of the Oligonucleotides note probes with 5′-extension (5 nt)

probes with 5′-extension (8 nt)

probes with 5′-extension (12 nt)

probes without 5′-extension (0 nt) probes with 3′-extension (5 nt)

5′-phosphorylated probes with 5′-extension (5 nt)

sequence (5′→ 3′)α TGA GGC TAA ACA ACT GCT TTT GCA GTT GTT TAG CCT CAG CAG TTG TTT AGT TTT CTA AAC AAC TGC TGA GGA TAC TAA ACA ACT GCT TTT GCA GTT GTT TAG TAT CCT CAG CAG TTG TTT AGT TTT CTA AAC AAC TGC TGA GGA TAA ACGCTA AAC AAC TGC TTT TGC AGT TGT TTA G CGT TTA TCC TCA GCA GTT GTT TAG TTT TCT AAA CAA CTG C GCA GTT GTT TAG TTT TCT AAA CAA CTG C CTA AAC AAC TGC TTT TGC AGT TGT TTA GTG AGG GCA GTT GTT TAG TTT TCT AAA CAA CTG CCC TCA PO4 TGA GGC TAA ACA ACT GCT TTT GCA GTT GTT TAG PO4 CCT CAG CAG TTG TTT AGT TTT CTA AAC AAC TGC

α

The italic letters indicate the extension region of hairpin probes, and the underlined letters represent the complementary region within the hairpin probes.

nucleosides triphosphate including cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), and deoxycytidine triphosphate (dCTP), and E. coli ligase (60 U/μL) were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The T4 polynucleotide kinase and 10× CutSmart buffer (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(Ac)2, 100 μg/mL BSA, pH 7.9) were purchased from New England Biolabs (Ipswich, MA). Nicotinamide adenine dinucleotide (NAD+), phosphoenolpyruvic acid monosodium salt hydrate (PEP), pyruvate kinase from rabbit muscle (PK), 8482

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Scheme 1. Principle of Label-Free Bioluminescent Sensor for Real-Time Monitoring of the Activity of Polynucleotide Kinase (PNK) Based on the Intramolecular Ligation of Two Hairpin Probes

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RESULTS AND DISCUSSION Principle of Bioluminescence Monitoring of the PNK Activity. The label-free bioluminescent sensor for PNK assay is developed on the basis of PNK-dependent DNA phosphorylation reaction, which can trigger the DNA ligation reaction and generate the ligated byproduct (AMP)-dependent bioluminescence signal (Scheme 1). We designed two hairpin probes with the complementary base sequences at their 5′ protruding termini. Because of the lack of 5′-phosphoryl (5′PO4) group, no ligation reaction occurs between them in the absence of PNK even though they can hybridize with each other to form a nicked double-stranded DNA. Upon the addition of PNK, both 5′-ends of two probes are phosphorylated, and their subsequent hybridization makes the 5′-PO4 group of one probe adjacent to the 3′-hydroxyl (3′-OH) group of the other probe, generating two ligatable sites. The DNA ligation reaction is initiated by the catalyzation of E. coli ligase in the presence of cofactor NAD+,38 releasing the byproduct of AMP to trigger the enzymatic cascade conversion for the generation of distinct bioluminescence signal.42 The whole signal production involves the conversion of AMP → ADP → ATP in the presence of AK, PEP, PK, and dCTP as well as the initiation of firefly luciferase−luciferin system by the converted ATP for the generation of bioluminescence signal (see Supporting Information, Figure S1A). Importantly, the generated bioluminescence signal is stable due to the reversible conversion between AMP and ATP,43−45 enabling it possible to continuously monitor the stepwise process of DNA phosphorylation-ligation reaction for label-free real-time monitoring of the PNK activity. To obtain the maximum reaction efficiencies for both phosphorylation and ligation, unique design of DNA probe is required. First, because the PNK is incapable of catalyzing the phosphorylation of dsDNA at an intramolecular ligation site46 (Supporting Information Figure S1B), the intermolecular ligation of two probes is adopted (Scheme 1) despite the higher ligation efficiency at the intramolecular site. Second, the hairpin conformation (Scheme 1) ensures the complete ligation reaction between the phosphorylated terminals. It should be noted that in the hybridized dsDNA conformation (Supporting Information Figure S1C), the two 5′-phosphorylated dsDNAs with blunt end cannot be ligated by E. coli ligase.47 In addition, because much shorter DNA sequence is required for the intramolecular hairpin hybridization than for intermolecular two-ssDNA hybridization,48 the synthesis of hairpin probe is much more cost-effective. Validation by Gel Electrophoresis. To validate the feasibility of the proposed method, we analyzed the reaction products under various phosphorylation conditions using a 15%

native polyacrylamide gel electrophoresis (PAGE). As shown in Figure 1, a distinct band of ligated DNA probe (the molecular

Figure 1. Native PAGE analysis of the products obtained from DNA phosphorylation-ligation reaction under various phosphorylation conditions.

weight is the sum of two single hairpin probes) is observed in the presence of PNK, ATP donor, ligase, and NAD+ cofactor (Figure 1, lane 2), suggesting that the probes with 5′-OH have been phosphorylated and ligated by the catalyzation of ligase. However, the band of the ligated DNA is not observed in the absence of PNK (Figure 1, lane 1), phosphate donor (Figure 1, lane 9), ligase (Figure 1, lane 3), and NAD+ (Figure 1, lane 4). These results confirm that the ligation of DNA probes can occur only after the phosphorylation, suggesting that it is feasible to monitor the PNK activity through monitoring the DNA probe ligation triggered by the PNK phosphorylation. When the phosphate donor of ATP is substituted by dCTP, similar results are observed (Figure 1, lanes 5−8), consistent with the report that other nucleosides triphosphate (such as GTP, UTP, CTP) can serve as the equally effective phosphate donor like ATP.2 Alternative to the generally used ATP, the use of other phosphate donors can efficiently eliminate the background signal in the bioluminescence measurement, which usually relies on the ATP as the signal reporter. Bioluminescence Assay. We further measured the ligated byproducts of AMP using a bioluminescence signal readout based on the coupled enzymatic conversion reactions (see Supporting Information, Figure S1A). With the addition of the AMP-to-ATP conversion buffer and the ATP detection buffer to the different reaction products, an enhanced bioluminescence signal is observed in the presence of all components (Figure 2A, red line), while the lack of any component results in a relatively low bioluminescence signal (Figure 2A, except for red line), suggesting that the ligation occurs only when probes are phosphorylated by PNK, consistent with the electrophoresis 8483

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bioluminescence signal (Figure 3A). Moreover, the bioluminescence signal increases with the dCTP concentration

Figure 2. Bioluminescence monitoring the byproduct (AMP) of phosphorylation-ligation reaction using either ATP (A) or dCTP (B) as the phosphate donor under various experimental conditions. RLU represents the relative light units.

Figure 3. Influence of phosphate donors (A) and the dCTP concentration (B) upon the measured bioluminescence signal. I0 and I represent the measured bioluminescence signal in the absence and in the presence of PNK, respectively. Error bars show the standard deviation of three experiments.

results (Figure 1). In addition, the cycling utilization of AMP leads to a stable bioluminescence signal (Figure 2A, red line). However, a fairly high background bioluminescence signal is still observed in the absence of PNK (Figure 2A, black line) due to the presence of a large number of unreacted ATP donors, which might initiate the firefly luciferase−luciferin system as well. Although the reduction of ATP dosage may partly reduce the background bioluminescence signal, the background interference cannot be removed completely due to the conversion of ATP to ADP2 and the participation of ADP in the conversion process of AMP-to-ATP.42 To eliminate the background signal, we replaced the extensively used ATP with dCTP as the donor. The dCTP has been proved to be as effective as ATP as a phosphate donor by electrophoresis analysis (Figure 1), but less effective than ATP substrate recognized by the bioluminescence system of firefly luciferase− luciferin.49 With dCTP as a phosphate donor, a significant PNK-dependent signal enhancement is observed in the presence of PNK, but no background bioluminescence signal is observed in the absence of PNK (Figure 2B), suggesting the dCTP donor is more suitable for the bioluminescence detection of PNK than the ATP donor. Optimization of Experimental Conditions. To improve the performance efficiency of PNK assay, we investigated the effect of various nucleosides triphosphate including GTP, UTP, dCTP, and CTP (except ATP) upon the measured bioluminescence signal. Because of the different background emission49 (see Supporting Information, Figure S2) and the different velocity of phosphorylation reaction2 induced by the four phosphate donors, only dCTP produces the highest

from 0 to 0.6 mM, followed by a slight decrease beyond the concentration of 0.6 mM (Figure 3B) due to the inhibition of both the phosphorylation efficiency and the luciferase− luciferin-based bioluminescence emission by high-concentration dCTP.19 Therefore, 0.6 mM dCTP is used in the following bioluminescence experiments. For PNK assay, the DNA probe is usually used as the substrate of PNK and acts as the phosphate acceptor as well. A wide range of nucleic acid substrates including even the small molecule of 3′-phosphate mononucleotide can accept the phosphoric group of the donor to get phosphorylated by PNK at their 5′ terminus.3 However, the phosphorylation efficiency may vary with the conformation of DNA probes50,51 and affect the subsequent ligation reaction. To maximize the efficiency of both phosphorylation and ligation, we designed the hairpin probes with 5′-extension (Scheme 1). Because of the presence of the hybridization equilibrium between the two hairpin probes, in theory, the shorter extension may facilitate the ssDNA conformation, while the longer extension may facilitate the dsDNA formation. As a result, the single probe is the preferred substrate of PNK, while the hybrids of them are preferred by the ligase (Figure 4A). Therefore, the extension length of 5′-end plays an important role in the PNK assay and should be optimized experimentally. When extending the 5′end length of the probes from 0 to 5 nt, the bioluminescence signal increases significantly (Figure 4B) due to the low ligation efficiency for the DNA probe with a blunt end47 and the high ligation efficiency for the DNA probe with a 5 nt-adhesive end.52 However, further extension of the 5′-end to 8 nt and 12 8484

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Figure 4. (A) Schematic illustration showing the effect of 5′protruding length of probes on the phosphorylation-ligation reaction. (B) The variance of bioluminescence signal in response to different 5′protruding length. I0 and I represent the measured bioluminescence intensity in the absence and in the presence of PNK, respectively. Error bars show the standard deviation of three experiments.

Figure 5. (A) Variance of bioluminescence signal in response to different amounts of PNK. (B) Variance of initial phosphorylation velocity (V) with the PNK concentration (C). Inset shows the linear relationship between the initial phosphorylation velocity (V) and the PNK concentration (C). Error bars show the standard deviation of three experiments.

nt leads to the decrease of bioluminescence signal (Figure 4B) due to the inhibition of phosphorylation efficiency by the long extension (Figure 4A). Therefore, 5 nt extension at the 5′-end of the probe is used in the following bioluminescence experiments. Bioluminescence Real-Time Monitoring of PNK Activity. Under the optimal conditions, we monitored the variance of bioluminescence with the PNK concentration in real time. As shown in Figure 5A, the bioluminescence signal increases with the increasing PNK concentration. The bioluminescence signal reaches equilibrium within 30 min, slightly longer than that required for the real-time fluorescence measurement (15 min)19 due to the lower reaction temperature used in this research (25 °C) than 37 °C used in the real-time fluorescence measurement.19 It should be noted that the optimal temperature of bioluminescence assay is limited to 22− 28 °C because of the low thermostability of firefly luciferase (it tends to be rapidly inactivated at the temperature of >30 °C).53 With the addition of glycine betaine to increase the stability of luciferase,54 the coupled enzymatic reactions-based PNK assay may be performed at the elevated temperatures. As shown in the inset of Figure 5B, the initial rate shows a linear correlation with the PNK concentration. The linear relationship can be described as V = 635.79C + 3.97 with a correlation coefficient of 0.9948, where V is the initial velocity and C is the PNK concentration (U/mL). The detection limit is calculated to be 0.004 U/mL based on 3 times standard deviation over the blank response. In comparison with the established approaches

for PNK assay (see Supporting Information, Table S1), our method has significant advantages of being simple, rapid, and cost-effective. To the best of our knowledge, this is the first demonstration of label-free and real-time monitoring PNK activity. In addition, as compared to the frequently used fluorescent method, our method does not require the external excitation, and is free from the autofluorescence interference of biomolecules matrix. Kinetic Analysis. We further employed this bioluminescent sensor to monitor the phosphorylation reactions in response to various DNA substrates. In the presence of PNK, the phosphorylation rate increases with the increasing concentration of DNA substrate with 5′-protruding from 0 to 80 nM (see Supporting Information, Figure S3A), but the background signal remains unchanged in the absence of PNK (see Supporting Information, Figure S3B). Similar results were observed for DNA substrate with 5′-recessed (see Supporting Information, Figure S3C and D), but the phosphorylation rate increases more slowly for DNA substrate with 5′-recessed than that for DNA substrate with 5′-protruding (see Supporting Information, Figure S3A and C), consistent with that dsDNA with 5′-protruding exhibits much higher phosphorylation efficiency than those with other conformations.55 The kinetics of phosphorylation reaction follows the Michaelis−Menten equation (Figure 6). The Michaelis−Menten constant (Km) 8485

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Figure 6. Analysis of Michaelis−Menten parameters using the DNA probe substrate with 5′-protruding (A) and 5′-recessed (B), respectively. Error bars show the standard deviation of three experiments.

and maximum initial velocity (Vmax) are measured to be 39.5 nM and 2643.1 min−1 for DNA substrate with 5′-protruding (Figure 6A), and 63.9 nM and 628.6 min−1 for DNA substrate with 5′-recessed (Figure 6B). These results demonstrate that the proposed method can be used for kinetic analysis. Inhibition Assay and Real Sample Analysis. We further investigated the effect of two model inhibitors including (NH4)2SO4 and Na2HPO4 upon the phosphorylation reaction. Because high-concentration (NH4)2SO4 and Na2HPO4 exhibit a significant inhibition effect on the subsequent enzymatic reactions including ligation, conversion, and detection (see Supporting Information, Figure S4), we diluted the phosphorylated DNA to eliminate the detrimental effect of inhibitors. With the increase of inhibitor concentration, the bioluminescence signal remains unchanged using the 5′phosphorylated DNA probe (Figure 7A,B, curves a), but decreases using the DNA probes without 5′-phosphoryl group (Figure 7A,B, curves b), suggesting the specific inhibition effect of (NH4)2SO4 (Figure 7A) and Na2HPO4 (Figure 7B) upon the PNK activity. The IC50 value of (NH4)2SO4 and Na2HPO4 is calculated to be 9.88 and 24 mM, respectively, consistent with the reported IC50 values of 10 mM for (NH4)2SO4 and 20 mM for Na2HPO4,22 suggesting the feasibility of the proposed method for the screening of PNK inhibitors. To demonstrate the capability of the proposed method for real sample analysis, we measured the PNK activity of the extract nucleoprotein in the human embryonic kidney cell line (HEK293T). The bioluminescence signal increases with time upon the addition of cell nucleoprotein (Figure 7C, curve a). In contrast, the bioluminescence signal remains unchanged in the nucleoprotein denatured by heating treatment (Figure 7C, curve b), while the further addition of PNK into the extracted nucleoprotein results in an enhanced bioluminescence signal (Figure 7C, curve c). It should be noted that the background

Figure 7. (A and B) Inhibition effect of (NH4)2SO4 (A) and Na2HPO4 (B) upon the PNK activity. Curves a and b represent the measured PNK activity using the DNA probes with and without 5′phosphate group, respectively. (C) Analysis of PNK activity in real samples: a, the extracted nucleoprotein; b, the extracted nucleoprotein denatured by heating treatment; c, the extracted nucleoprotein with the addition of PNK.

emission of denatured nucleoprotein is higher than that of the pure buffer due to the presence of a small amount of AMP, ADP, and ATP in the extraction solution of protein. According to the standard work curve (see Supporting Information, Figure S5), the PNK activity of extracted nucleoprotein is measured to be 0.056 U/mL. Furthermore, we measured the cell samples spiked with 0.2, 0.4, and 0.6 U/mL PNK, and obtained the quantitative recovery in the range from 96.8% to 104.0% (see Supporting Information, Table S2).

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CONCLUSIONS In summary, we have developed a label-free bioluminescent sensor for real-time monitoring of the PNK activity. This bioluminescent sensor is constructed on the basis of PNKdependent addition of 5′-phosphates to the oligonucleotides for the initiation of ligation reaction and the subsequent release of AMP for the generation of self-illuminating light emission. To the best of our knowledge, this is the first demonstration of label-free and real-time monitoring PNK activity. The proposed 8486

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method is much simpler, label-free, and cost-effective, and it can be used for quantitative, kinetic, and inhibition assay, holding great potential for further application in biomedical research and drug development.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figures S1−S5 and Tables S1−S2. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86 931 7971276. Fax: +86 931 7971323. E-mail: luxq@ nwnu.edu.cn. *Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Natural Science Foundation of China (Grant nos. 21325523, 21327005, and 21175108), the Program for Changjiang Scholars and Innovative Research Team, Ministry of Education of China (Grant no. IRTI283), the Award for the Hundred Talent Program of the Chinese Academy of Sciences, and the Program of Innovative Research Group of Gansu Province of China (Grant no. 1210RJIA001).

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