Single-Molecule Detection of Polynucleotide Kinase Based on

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Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticle Li-juan Wang, Qianyi Zhang, Bo Tang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 6, 2017

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Analytical Chemistry

Single-Molecule Detection of Polynucleotide Kinase Based on Phosphorylation-Directed Recovery of Fluorescence Quenched by Au Nanoparticle ﹟,†

Li-juan Wang, ﹟

﹟

﹟

Qianyi Zhang, ‡,† Bo Tang, ,* and Chun-yang Zhang ,*

College of Chemistry, Chemical Engineering and Materials Science, Collaborative

Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China ‡

Nantou High School Shenzhen, Shenzhen, 518052, China

* Corresponding author. Tel.: +86 0531-86186033; Fax: +86 0531-82615258. E-mail: [email protected].

Tel.:

+86

0531-86180010;

Fax: +86

[email protected].

1

ACS Paragon Plus Environment

0531-86180017;


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Abstract: 5′-Polynucleotide kinase such as T4 polynucleotide kinase (T4 PNK) may catalyze the phosphorylation of 5′-hydroxyl termini in nucleic acids, playing a crucial role in DNA replication, DNA recombination and DNA damage repair. Here, we demonstrate for the first time single-molecule detection of PNK based on phosphorylation-directed recovery of fluorescence quenched by Au nanoparticle (AuNP) in combination with lambda exonuclease-mediated cleavage reaction. In the presence of PNK, the γ-phosphate group from adenosine triphosphate (ATP) is transferred to 5′-hydroxyl terminus, resulting in 5′-phosphorylation of hairpin probe. The phosphorylated hairpin probes may function as the substrates of lambda exonuclease and enable the removal of 5′ mononucleotides from the stem, leading to the unfolding of hairpin structure and the formation of binding probes. The resultant binding probes may specifically hybridize with the AuNP-modified capture probes, forming double-strand DNA (dsDNA) duplexes with 5′-phosphate groups as the substrates of lambda exonuclease and subsequently leading to the cleavage of capture probes and the liberation of Cy5 molecules and the binding probes. The released binding probes may further hybridize with new capture probes, inducing cycles of digestion-release-hybridization and consequently the release of numerous Cy5 molecules. Through simply monitoring Cy5 molecules with total internal reflection fluorescence (TIRF)-based imaging, PNK activity can be quantitatively measured. This assay is very sensitive with a limit of detection of 9.77 × 10−8 U/µL, and it may be further used to screen the PNK inhibitors and measure PNK in cancer cell extracts.

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Introduction. Various exogenous and endogenous agents including ionizing radiation, chemicals and nucleases may cause the hydroxylation of 5′ termini in nucleic acids, resulting in DNA damage and genomic instability.1-4 The 5′-Polynucleotide kinase (PNK) is responsible for the repair of 5′-hydroxyl radical of nucleic acids through transferring the γ-phosphate group from adenosine triphosphate (ATP) to the 5′-hydroxyl group of oligonucleotides or nucleic acids.5 Malfunction of PNK may cause the deregulation of many cellular activities (e.g., nucleic acid metabolism, DNA replication, DNA recombination and DNA repair during strand damage),6-9 and eventually induces a variety of human diseases including loom’s syndrome, Werner syndrome and Rothmund-Thomson syndrome.10 Additionally, PNK is becoming a promising therapeutic target because of the positive effect of 5′-polynucleotide kinase inhibitors in the radio therapy of somatic cancers.11,12 Given the important roles of PNK in biology and drug development, sensitive detection of PNK activity is of great importance to the fundamental biochemical research, clinic diagnosis and drug discovery. Traditional methods for PNK assay are mainly based on radical isotope

32

P-labeling,

polyacrylamide gel electrophoresis (PAGE) and autoradiography.13-17 But they suffer from several intrinsic drawbacks, including radioactive hazards, complicated manipulation, time-consuming and labor-intensive procedures, greatly limiting their broad applications. Alternatively, a variety of new approaches have been developed for PNK assay, including colorimetric,18 electrochemical,19,20 chemiluminescent,21,22 bioluminescent,23 and fluorescent assays.9,24-26 Among them, the fluorescent method attracts more and more attention due to its distinct advantages of convenience, high throughput and improved sensitivity. Tang combined 3



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molecular beacons with ligation reaction for sensitive monitoring of phosphorylation process.9 Song demonstrated the use of fluorophore-labeled hairpin DNA probe for real-time detection of PNK activity.24 Hou employed bimolecular beacon-based signal amplification to sensitively detect PNK activity and screen PNK inhibitors.25 Lin used graphene oxide as a superquencher to measure PNK activity.26 Huang developed a fluorescence polarization-based biosensor for PNK assay.27 Although these methods work well for PNK assay, the further improvement of detection sensitivity still remain a great challenge, and especially the development of simple, rapid and sensitive methods is highly desirable. Due to their unique optoelectronic properties, excellent biocompatibility, high catalytic activity and potential noncytotoxicity,28-31 Au nanoparticles (AuNPs) have become the focus of extensive research, including biochemical analysis, bioelectronics, biomolecule labeling, nano biochips and nano biosensors.32,33 Taking advantage of extremely high extinction coefficients (108-109) of their surface plasmon resonance absorption and the strong distance-dependent optical properties among the inter particles,34,35 AuNPs have been widely used as the colorimetric materials to fabricate nanobiosensors.30,36,37 Especially, AuNPs may function as the superquenchers to efficiently quench the fluorescence of various dyes,38,39 and they have been used to construct nanosensors for the detection of DNAs,40 proteins41 and small molecules.42 However, these AuNP-based nanosensors inevitably suffer from a high background signal, because the AuNPs can only quench the conventional fluorophores in a limited distance of ~10 nm.43 To eliminate the high background signal and improve the detection sensitivity, we may alternatively monitor the fluorophores dissociated from the AuNPs by introducing lambda exonuclease-mediated cleavage reaction and total internal 4


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reflection fluorescence (TIRF)-based single-molecule imaging. The lambda exonuclease is a highly processive 5′-3′ enzyme which can remove 5′ mononucleotides from the dsDNA duplex to generate single-stranded DNA (ssDNA) and mononucleotides. The TIRF-based imaging has distinct advantages of high resolution, low sample consumption and high signal-to-noise ratio, and have been successfully applied for sensitive detection of DNAs,44 miRNAs45 and enzymes at the single-molecule level.46-51 In this research, we demonstrate for the

first

time

single-molecule

detection

of

polynucleotide

kinase

based

on

phosphorylation-directed recovery of fluorescence quenched by Au nanoparticle in combination with lambda exonuclease-mediated cleavage reaction. This method can sensitively detect PNK with a limit of detection of as low as 9.77 × 10−8 U/µL, and it exhibits a large dynamic range of 4 orders of magnitude from 1 × 10−7 to 1 × 10−3 U/µL. Furthermore, it can be applied for the screening of PNK inhibitors and the measurement of PNK in the cancer cell extracts.

EXPERIMENTAL SECTION Chemicals and Materials. All HPLC-purified DNA oligonucleotides (Table 1) were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). The T4 polynucleotide kinase and 10× T4 polynucleotide kinase reaction buffer (700 mM trizma hydrochloride (Tris-HCl), 50 mM DL-Dithiothreitol (DTT), 100 mM magnesium chloride (MgCl2), pH 7.6), lambda exonuclease, 10× lambda exonuclease reaction buffer (670 mM glycine-potassium hydroxide (KOH), 25 mM MgCl2, 500 µg/mL bovine serum albumin (BSA), pH 9.4), and adenosine 5



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5'-triphosphate (ATP) were obtained from New England Biolabs (Beverly, MA, USA). Adenylate kinase (AK), adenosine diphosphate (ADP), ammonium sulfate (NH4)2SO4), glycine, KOH, MgCl2, D-glucose, glucose oxidase, trolox, BSA and catalase were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). The Au nanoparticles were bought from Nanocs, Inc. (New York, NY, USA). Ultrapure water obtained from a Millipore filtration system was used in all experiments.

Table 1. The sequences of Oligonucleotidesα note hairpin probe

sequence (5'-3') OH-CAG CAG TTG TTT AGA TTT TTT TAT CTA AAC AAC TGC TG

capture probe

P-CAG CAG T(Cy5)TG TTT TT-biotin

binding probe

TTT TTT CTA AAC AAC TGC TG

α

In hairpin probe, the “OH” denotes the hydroxyl group modified at the 5′ end, and the

underlined regions signify the complementary sequences. In capture probe, the “P” and “biotin” denote the phosphate group and biotin molecule, respectively, and the underlined base “T” is modified with Cy5.

Preparation of Capture Probe-Modified AuNPs. The capture probe (5.9 nmol) was added to 1 mL of AuNP solution (5.7 × 1012 particles/mL), and incubated at room temperature for 16 h, followed by standing at room temperature in PBS buffer (10 mM phosphate (NaH2PO4/Na2HPO4), 0.1 M NaCl, pH 7.0) for 36 h. The AuNPs 6


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were centrifuged three times to remove the unbound capture probes, and then resuspended in 250 µL of PBS buffer (10 mM phosphate, 0.1 M NaCl, pH 7.0) and stored at 4 °C. In the capture probe-modified AuNP solution, the concentration of capture probe was estimated to be 18.5 µM (see Supporting Information, Figure S1).

PNK-Catalyzed

Phosphorylation

and

Lambda

Exonuclease-Mediated

Cleavage

Reaction-Induced Recovery of Fluorescence Quenched by AuNPs. All oligonucleotides were dissolved in 1× Tris-EDTA buffer. The hairpin probes were diluted to 1 µM and incubated in 10 mM Tris-HCl (pH 8.0) and 1.5 mM MgCl2 for 4 min at 95 °C for denaturation, and then cooled to the room temperature to enable the formation of hairpin structure. The 2.5 µL of hairpin probes was added to the phosphorylation reaction system (50 µL) consisting of various-concentration PNK, 0.6 mM ATP and 5 µL of 10× T4 polynucleotide kinase reaction buffer, and incubated at 37 °C for 30 min. After phosphorylation reaction, 10 U lambda exonuclease, 5 µL of 10× lambda exonuclease reaction buffer and 1.4 µL of capture probe-modified AuNPs were added into the reaction system and incubated for 40 min at 37 °C to perform lambda exonuclease-mediated cyclic cleavage reaction-induced recovery of fluorescence quenched by AuNPs.

Measurements of Fluorescence Spectra and Electrophoresis Analysis. The reaction products (25 µL) were diluted to 60 µL with ultrapure water for the measurement of fluorescence spectra by a HitachiF-7000 fluorescence spectrophotometer (Tokyo, Japan). The emission spectra were recorded at a scan rate of 2 nm/s with an excitation wavelength of 7



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646 nm, and the fluorescence intensity at 670 nm was recorded for data analysis. To analyze the reaction products, 12% nondenaturating polyacrylamide gel electrophoresis (PAGE) was performed in 1× TBE buffer (9 mM Tris-HCl, 0.2 mM EDTA, 9 mM boric acid, pH 7.9) at room temperature. After electrophoresis at 110 V for 30 min, the gel was stained by a silver staining kit (Tiandz Inc., Beijing, China).

TIRF-Based Single-Molecule Detection The reaction products were diluted 1000-fold with the imaging buffer (0.4% (w/v) D-glucose, 1 mg/mL glucose oxidase, 50 µg/mL BSA, 67 mM glycine-KOH, 0.04% mg/mL catalase, 2.5 mM MgCl2, 1 mg/mL trolox, pH 9.4). The 10 µL of samples was used for TIRF imaging. The Cy5 molecules were excited by a sapphire 640 nm laser (Coherent, USA), and the Cy5 signals were collected by Andor Ixon DU897 EMCCD. The image J software was used for the counting of Cy5 emission.

Inhibition Assay. For PNK inhibition assay, ADP and (NH4)2SO4 at various concentration were added, respectively, into the reaction system, followed by single-molecule detection. The relative

activities (RA) of PNK were calculated according to the following equation: RA =

Ni − No , Nt − No

where No represents the Cy5 counts when PNK is absent, Nt represents the Cy5 counts when 0.01 U/µL PNK is present, and Ni represents the Cy5 counts when both 0.01 U/µL PNK and the inhibitor are present. The IC50 was estimated on the basis of the fitting curve of RA against the inhibitor concentration. 8


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Preparation of Cell Extracts. The human embryonic kidney (HEK293T) cells were used for the preparation of cell extracts. According to the manufacturer’s protocol, the cell extracts were obtained with nucleoprotein extract kit (BSP001, Sangon Biotech, Shanghai, China).

RESULTS AND DISCUSSION Principle of PNK Assay. In this research, we used T4 polynucleotide kinase (T4 PNK) as the model target.52-54 This assay consists of three consecutive steps: (1) PNK-actuated addition of phosphate group to the 5′-hydroxyl termini of the hairpin probe; (2) lambda exonuclease-mediated cyclic release of Cy5 from the Cy5-capture probe-AuNP nanostructure; and (3) TIRF-based single-molecule detection. As shown in Scheme 1, the hairpin probe is designed with a loop and a stem. The loop structure (Scheme 1, blue color) is designed to prevent the stem from being processively cleaved by lambda exonuclease. The stem contains two complementary strands, i.e., the upper strand (Scheme 1, black color) and the down strand (Scheme 1, red color). The 5′ end of the upper strand is modified with a hydroxyl group for PNK-actuated phosphorylation reaction, and the down strand is the partial complementary sequence of the capture probe. The capture probe is modified with a phosphate group (PO4) at the 5′ end and a sulfhydryl (SH) group at the 3′ end, with a fluorophore (Cy5) being modified on the adenine (A) base that is 6 bases away from the 5′ end. The capture probes may be coated on the surface of AuNPs through S-Au covalent binding to form the Cy5-capture probe-AuNP nanostructures. In the presence of PNK, the 9



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γ-phosphate residue from adenosine triphosphate (ATP) may be transferred to the 5′-hydroxyl termini, resulting in the addition of 5′-phosphate group to the hairpin probe. The 5′-phosphorylated hairpin probes may function as the substrates for lambda exonuclease and are specifically cleaved through catalyzing the removal of 5′-mononucleotides one by one from the dsDNA duplexes, leading to the unfolding of hairpin structure and the formation of binding probes. With the addition of capture probe-modified AuNPs, the binding probes may hybridize with the capture probes to form the dsDNA duplexes. The resultant dsDNA duplexes with 5′-phosphate groups may function as the substrates for lambda exonuclease, resulting in the cleavage of capture probes from 5´-phosphate ends to 3´-hydroxyl ends and eventually the release of Cy5 molecules and binding probes. The released binding probes may hybridize with the new capture probes in the Cy5-capture probe-AuNP nanostructures, generating the cycles of cleavage-release-hybridization and consequently the release of a large number of Cy5 molecules into the solution. Therefore, the PNK activity can be quantitatively and accurately measured by simply counting the released Cy5 molecules. On the contrary, the PNK-actuated phosphorylation reaction cannot be initiated in the absence of PNK, and neither the cleavage of capture probes nor the release of Cy5 molecules into the solution may occur, and thus no Cy5 signal can be observed. Due to the ultrahigh quenching capability of AuNPs, the efficient cleavage of dsDNA with 5′-phosphoryl termini by lambda exonuclease and the high sensitivity of TIRF-based single-molecule detection, this assay can be used to sensitively measure PNK activity.

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Scheme 1. Scheme illustration of single-molecule detection of PNK based on lambda exonuclease-mediated cyclic cleavage reaction-induced recovery of fluorescence quenched by AuNPs. This assay involves three consecutive steps: (1) PNK-actuated phosphorylation reaction, (2) lambda exonuclease-mediated cyclic cleavage reaction-induced recovery of fluorescence quenched by AuNPs, and (3) TIRF-based single-molecule detection.

Validation of PNK Assay. We used nondenaturating PAGE to analyze the reaction products. One 38-nt band is observed (Figure 1A, lane 1) in the absence of PNK, indicating that the DNA substrate with 5′-hydroxyl termini cannot be cleaved by lambda exonuclease. While in the presence of PNK, two distinct bands of 38 nt and 22 nt are observed (Figure 1A, lane 2), suggesting that PNK enables the addition of γ-phosphate groups from ATP to the 5′-hydroxyl termini of hairpin probes which are subsequently cleaved by lambda exonuclease to generate a 22-nt product. The obtained 22-nt product is confirmed by the positive control with synthetic binding probes (Figure 1A, lane 3). We further performed fluorescence 11



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measurement to verify the feasibility of the proposed assay. The capture probe-modified AuNPs are incubated with the reaction products and then subjected to lambda exonuclease digestion prior to the fluorescence measurement. No obvious Cy5 signal is detected without PNK (Figure 1B, black curve), indicating that no Cy5 molecule is released from the Cy5-capture probe-AuNP nanostructures in the absence of PNK. While in the presence of PNK, a distinct Cy5 signal is observed (Figure 1B, red curve), suggesting that PNK can actuate the phosphorylation of 5′-hydroxyl termini in DNA substrates to initiate the lambda exonuclease-mediated cyclic cleavage reaction for the release of abundant Cy5 molecules into the solution. Both the gel electrophoresis (Figure 1A) and fluorescence measurements (Figure 1B) clearly demonstrate that PNK can successfully transfer the γ-phosphate residue from ATP to the 5′-hydroxyl termini of dsDNA strands and initiate the subsequent lambda exonuclease-mediated

cyclic release of Cy5 from the Cy5-capture

probe-AuNP

nanostructures. The AuNPs can function as the highly efficient fluorescence quenchers over long distances due to their large size, no defined dipole moment, large absorption cross section and radiative rate suppression.43,55 To enable Cy5 to be efficiently quenched by AuNPs, the effective separation distance between the AuNP and Cy5 must be met. In theory, the distance between two adjacent bases is 0.34 nm for dsDNA,56 and the distance between AuNP and Cy5 in the Cy5-capture probe-AuNP nanostructure is calculated to be 7.38 nm (the diameter of the AuNP is 10 nm), within the efficient quenching range of AuNP (the effective quenching distance between the AuNP and Cy5 is reported to be 2.2-16.6 nm).43 Thus, AuNPs can efficiently quench the fluorescence of Cy5 in the Cy5-capture probe-AuNP nanostructures. In the 12


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presence of PNK, PNK can actuate the phosphorylation of 5′-hydroxyl termini in DNA substrates to initiate the lambda exonuclease-mediated cyclic cleavage reaction, releasing a large number of Cy5 from the Cy5-capture probe-AuNP nanostructures and generating an enhanced fluorescence signal. To demonstrate the proof of concept, we monitored the change of Cy5 fluorescence intensity with the PNK concentration. The Cy5 fluorescence intensity enhances with the increasing PNK concentration (Figure 2A). In the logarithm scales, the Cy5 fluorescence intensity exhibits a linear correlation with the PNK concentration from 1 × 10−6 to 1 × 10−3 U/µL (Figure 2B). The correlation equation is F = 221.4 log10 C + 1307.5 (R2 = 0.9000), where F is the Cy5 fluorescence intensity and C is the PNK concentration (U/µL). The limit of detection is calculated to be 1.26 × 10−6 U/µL. The sensitivity of this assay is comparable to those of reported colorimetric (0.0018 U/mL),18 electrochemical (0.003 U/mL)20 and bioluminescent assays (0.004 U/mL),23 and is 50 times higher than those of chemiluminescent (0.05 U/mL)21 and fluorescent assays (0.05 U/mL).26 These results clearly demonstrate that phosphorylation-directed recovery of fluorescence quenched by AuNP can be used for PNK assay.

Figure 1. (A) Analysis of the reaction products by nondenaturating PAGE. Lane 1 shows the products when PNK is absent. Lane 2 shows the products when PNK is present. Lanes 3 is the 13



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synthesized binding probe. (B) The Cy5 fluorescence spectra induced by 0.01 U/µL PNK (red curve) and the control without PNK (black curve).

Figure 2. (A) Change of Cy5 emission spectra with the PNK concentration. (B) Change of Cy5 fluorescence intensity at the wavelength of 646 nm with the PNK concentration. Error bars represent the standard deviation of 3 experiments.

TIRF-based Single-Molecule Detection for PNK Assay. TIRF-based fluorescence imaging57 was used for sensitive detection of Cy5 molecules released from the Cy5-capture probe-AuNP nanostructures as a result of PNK-actuated lambda exonuclease-mediated cyclic cleavage reaction (Figure 3A). In the control group without PNK, no Cy5 fluorescence signal is detected due to the absence of PNK-initiated lambda exonuclease-mediated cyclic cleavage reaction. While in the presence of PNK, a large number of Cy5 fluorescence signals are detected (Figure 3B) due to the release of abundant Cy5 molecules from the Cy5-capture probe-AuNP nanostructures induced by PNK-initiated lambda exonuclease-mediated cyclic cleavage reaction. Therefore, the measurement of Cy5 molecules released in the solution can be used for accurate quantification of PNK. Under the optimally experimental conditions (see Supporting Information, Figures S2-S4), we measured the Cy5 counts induced by 14


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different-concentration PNK (Figure 3C). The higher the PNK concentration, the more the Cy5 counts being detected. In the logarithm scales, the Cy5 counts exhibit a linear correlation with the PNK concentration in the range from 1 × 10−7 to 1 × 10−3 U/µL. The correlation equation is N = 55.7 log10 C + 397.2 (R2 = 0.9864), where C represents the PNK concentration (U/µL) and N represents the number of Cy5 counts. The limit of detection is calculated to be 9.89 × 10−8 U/µL. The sensitivity of this assay is 10 times higher than that of bulk measurement-based fluorescent assay (Figure 2B), and it has improved by 18.20-, 30.33-, 505.56-, 40.44- and 505.56-fold, respectively, compared with the DNAzyme-mediated colorimetric assay (0.0018 U/mL),18 TiO2 nanotube- and Au nanoparticle-based electrochemical assay (0.003 U/mL),20 G-quadruplex- and Ir(III) complex-mediated chemiluminescent assay (0.05 U/mL),21 enzymatic ligation-mediated bioluminescent assay (0.004 U/mL),23 and graphene oxide-based fluorescent assay (0.05 U/mL).26 The ultrahigh sensitivity may be attributed to (1) the high quenching capability of AuNPs, (2) the high efficiency of lambda exonuclease-mediated recycles of cleavage reaction, and (3) the high signal-to-noise ratio of TIRF-based single-molecule detection.

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Figure 3. (A-B) Single-molecule fluorescence image in the absence (A) and in the presence of PNK (B). The scale bar is 0.5 µm, and the PNK concentration is 0.01 U/µL. (C) Variance of Cy5 counts with the PNK concentration. In the logarithm scales, the Cy5 counts display a linear correlation with the PNK concentration (inset). Error bars show the standard deviation of 3 experiments.

Selectivity of PNK Assay. Because human nucleotide kinase is a superfamily, it is a great challenge to discriminate one kind of kinase from others.52 To evaluate the selectivity of this assay, we used adenylate kinase (AK) as the nonspecific interference. AK is a phosphotransferase enzyme that catalyzes the interconversion of adenine nucleotides (ATP, ADP, and AMP).58 Notably, AK enables the transfer of phosphate groups among ATP, ADP, and AMP, but it cannot transfer the phosphate groups to the 5′ termini of nucleic acids.58 In the control group with only reaction buffer (Figure 4, black column) and in the presence of AK (Figure 4, blue column), no Cy5 signal is detected. While in the presence of PNK, a significant Cy5 signal is detected (Figure 4, red column). The Cy5 counts measured in the 16


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presence of PNK is 75.0-fold and 53.6-fold more than those measured in the control and AK groups, respectively. These results demonstrate the excellent selectivity of this method for PNK assay.

Figure 4. Measurement of Cy5 counts induced by reaction buffer (black column), 0.01 U/µL PNK (red column), and 0.01 U/µL AK (blue column), respectively. Error bars represent the standard deviation of 3 experiments.

PNK Inhibition Assay. PNK is not only an important biomarker, but also a critical therapeutic target.12 Therefore, the screening of effective inhibitors is crucial to PNK-related cancer diagnosis and therapy. In this research, we used adenosine 5′-diphosphate sodium salt (ADP) and (NH4)2SO4 as the model inhibitors. The ADP is a noncompetitive inhibitor whose inhibition effect results from its reversible function in phosphorylation reaction.8 (NH4)2SO4 may suppress PNK activity through changing the structure of dsDNA substrate and the spatial conformation of PNK enzyme. 54 With a fixed amount of PNK, the change of Cy5 counts with the concentrations of ADP and (NH4)2SO4 was measured, respectively (Figure 5). The Cy5 counts decrease with the increasing concentrations of ADP (Figure 5A) and (NH4)2SO4 17



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(Figure 5B), respectively, indicating that both ADP and (NH4)2SO4 may efficiently inhibit PNK activity. The half-maximal inhibition values (IC50) of ADP and (NH4)2SO4 are calculated to be 1.45 mM and 2.99 mM, respectively, which are comparable to those reported IC50 values of ADP59 and (NH4)2SO4.23 These results demonstrated that this method can be used to screen the PNK inhibitors for drug development.

Figure 5. Change of the relative activity of PNK with the concentrations of ADP (A) and (NH4)2SO4 (B), respectively. The PNK concentration is 0.01 U/µL. Error bars show the standard deviations of 3 experiments.

Measurement of Cellular PNK. Accurate measurement of PNK in clinical samples is of great significance to biochemical research. As a proof-of-concept, we used human embryonic kidney cell line (HEK293T) as the model. The nucleoprotein was extracted from HEK293T cells, and the PNK concentration in the extracted nucleoprotein was accurately evaluated according to the calibration curve in Figure 3C. Figure 6 shows the change of Cy5 counts induced by PNK in the extracted nucleoprotein. In the logarithm scales, the Cy5 counts display a linear correlation with the PNK concentration in the range from 1 × 10−5 to 1 × 10−3 U/µL. The correlation equation is N 18


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= 62.2 log10 C + 321.0 with a correlation coefficient of 0.9795, where C represents the PNK concentration in the extracted nucleoprotein and N represents the number of Cy5 counts, respectively. The limit of detection is calculated to be 9.64 × 10−6 U/µL. This result indicates the capability of this method for accurate quantification of PNK in complex samples. Notably, only a few reported methods can detect but not quantify PNK activity in cell extracts and in the simulated complex samples,22,23,60 and most of the reported methods are not suitable for the detection of PNK activity in real samples.18-21,25,26

Figure 6. Change of Cy5 counts with the PNK concentration in the extracted nucleoprotein from HEK293T cells. Error bars represent the standard deviations of 3 experiments.

CONCLUSIONS We develop a new fluorescent method to sensitively detect PNK at the single-molecule level based on phosphorylation-directed recovery of fluorescence quenched by AuNPs. Taking advantage of the ultrahigh quenching capability of AuNPs, the high efficiency of lambda 19



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exonuclease-mediated catalytic recycle of cleavage reaction, and the high signal-to-noise ratio of TIRF-based single-molecule detection, this method can sensitively detect PNK with a limit of detection of 9.77 × 10−8 U/µL, and it exhibits a wide dynamic range from 1 × 10−7 to 1 × 10−3 U/µL, which

is superior to the reported colorimetric,18

electrochemical,20

chemiluminescent,21 fluorescent26 and bioluminescent assays.23 This method may accurately evaluate the inhibition effect of ADP and ammonium sulfate upon the phosphorylation, and quantitatively measure PNK activity in cancer cell extracts as well. Importantly, the proposed method may be used as a universal sensing platform to detect other polynucleotide kinase, and to simultaneously detect multiple nucleotide kinases through the design of appropriate DNA substrates and the selection of proper fluorophores paired with AuNPs.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge available free of charge on the ACS Publications website at DOI: Characterization of the capture probes-modified AuNPs; Optimization of the concentrations of ATP and lambda exonuclease, and the cleavage time of lambda exonuclease (PDF). AUTHOR INFORMATION Corresponding Author *Tel.: +86 0531-86186033. Fax: +86 0531-82615258. E-mail: [email protected]. . *Tel.: +86 0531-86180010. Fax: +86 0531-86180017. E-mail: [email protected]. Author Contributions 20


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† These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523 and 21527811), and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China.

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For TOC only

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Single-Molecule Detection of Polynucleotide Kinase Based on

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