Base-Excision-Repair-Induced Construction of a Single Quantum-Dot

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Base-Excision-Repair-Induced Construction of a Single QuantumDot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity Li-juan Wang,† Fei Ma,† 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 S Supporting Information *

ABSTRACT: DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually timeconsuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin−streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10−6 U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.

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syndrome.8 Dysregulation of OGG1 is also closely linked to multiple cancers including lung, breast, gastric, gallbladder, bladder, and orolaryngeal cancers.9−14 Therefore, the accurate and sensitive detection of DNA glycosylase activity is crucial to both biomedical research and clinical diagnosis. Conventional methods for DNA glycosylase assay include gel,15 radioactive method,16,17 mass spectrometry (MS),18 and high-performance liquid chromatography (HPLC). 19,20 Although these methods are efficient, they are usually timeand labor-consuming with low sensitivity and often involve hazardous radioactive materials, sophisticated instruments, and complicated procedures. To overcome these limitations, several new strategies including colorimetric21,22 and fluorescent methods23 have been developed in recent years. The

he maintenance of genomic integrity is of great importance to all organisms,1 but the genomes are constantly injured by various endogenous and environmental agents, resulting in many types of DNA lesions.1,2 If these DNA lesions are not repaired correctly, they may cause diverse human diseases.3 The repair of the damaged DNA involves a series of repair enzymes and several repair pathways such as nucleotide excision repair, double-strand DNA break repair, mismatch repair, and base excision repair.4 Among these repair pathways, base excision repair is the most versatile and it can repair various DNA damages resulting from alkylation, oxidation, and deamination.5 DNA glycosylase is the key repair enzyme of base excision repair.5,6 The abnormal level of DNA glycosylase in human cells may cause the malfunction of base excision repair and eventually various diseases. For example, high expression level of 8-oxoguanine DNA glycosylase (OGG1) is found in Parkinson patients.7 The knockout of Nei-like 1 (NEIL1) DNA glycosylase may lead to metabolic © XXXX American Chemical Society

Received: February 18, 2016 Accepted: July 12, 2016

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DOI: 10.1021/acs.analchem.6b00664 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

ration (California, U.S.A.). The silver staining kit was obtained from Tiandz, Inc. (Beijing, China). Detection of hOGG1 and Assembly of QD-DNA Substrate-Cy5 Nanostructure. The detection of hOGG1 activity involves three consecutive steps. First, all oligonucleotides were diluted with 10× Tris-EDTA buffer to prepare the stock solutions. The double-stranded DNA (dsDNA) substrates were prepared by incubating 1 μM sense strand with 1 μM antisense strand in a buffer containing 1.5 mM MgCl2 and 10 mM Tris−HCl (pH 8.0) at 95 °C for 2 min, followed by slowly cooling to room temperature. The obtained dsDNA substrates were stored at 4 °C for further use. Second, 2 μL of dsDNA substrates (1 μM) was added into 20 μL of reaction solution containing variable-concentration hOGG1, 10× NEB buffer 2 (500 mM NaCl, 100 mM Tris-HCl (pH 7.9), 100 mM MgCl2, 10 mM DTT), 1 U DNA polymerase β, polymerization reaction buffer (50 mM Tris-HCl (pH 8.7), 10 mM KCl, 10 mM MgCl2, 0.4 mg/mL BSA, 1 mM DTT, 15% (v/v) glycerol), 100 μg/mL BSA, 1 μM Cy5-labeled dGTP, 0.5 U APE1 and 10× NEB buffer 4 (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9), and the solution was then incubated at 37 °C in the dark for 30 min. Third, 20 μL of reaction products was mixed with 0.83 nM 605QDs in 100 μL of buffer (3 mM MgCl2, 100 mM Tris-HCl, 10 mM (NH4)2SO4, pH 8.0) for 10 min to form the 605QD-DNA-Cy5 complexes at room temperature. Steady-State Fluorescence Measurements and Gel Electrophoresis. The measurement of steady-state fluorescence was performed with a Hitachi F-4600 spectrometer (Kyoto, Japan). Before mixing with 605QDs, the products of hOGG1-induced base excision repair reaction were analyzed with 12% denaturating polyacrylamide gel electrophoresis (PAGE) in 1× TBE buffer (9 mM Tris-HCl, pH 7.9, 9 mM boric acid, 0.2 mM EDTA) at a 110 V constant voltage for 50 min at room temperature. The gel was stained with a silver staining kit (81104-1000, Tiandz Inc., Beijing, China) and visualized by a Kodak Image Station 4000 MM (Rochester, NY, U.S.A.). Single-Molecule Detection and Data Analysis. The reaction products were diluted 100-fold in imaging buffer (1 mg/mL glucose oxidase, 0.4% (w/v) D-glucose, 0.04% mg/mL catalase, 50 μg/mL BSA, 67 mM glycine-KOH, 1 mg/mL trolox, 2.5 mM MgCl2, pH 9.4). For TIRF imaging, 10 μL of samples was directly pipetted to the coverslips. A sapphire 488 nm laser (50 mW, Coherent, USA) was used to excite the 605QD. The photons from the 605QD and Cy5 were collected by a 100× objective (Olympus, Japan) and imaged with an exposure time of 100 ms by an Andor Ixon DU897 EMCCD. A region of interest (200 × 400 pixels) of the images was selected for Cy5 molecule counting by using image J software. Inhibition Assay. Variable-concentration CdCl2 was incubated with 0.1 U/μL hOGG1 at 37 °C for 15 min, followed by incubation with glycosylase reaction mixture at 37 °C for 60 min. The relative activity of hOGG1 (RA) was N −N measured according to RA = i o × 100%, where No is the

colorimetric assay enables the visualized detection of DNA glycosylase, but it involves either the complicated procedures for the preparation and modification of gold nanoparticle (AuNP)21 or the careful design of hairpin probe.22 To improve the sensitivity, an Exo III-assisted autonomous cycling cleavagebased fluorescent method is introduced,23 but the nonspecific digestion of Exo III may affect the assay specificity.24 Recently, the quantum dot (QD)-based fluorescence resonance energy transfer (FRET)25−33 technology with the integration of single-molecule detection34−36 enables the measurement of various biomolecules with improved sensitivity. Herein, we demonstrated the base-excision-repairinduced construction of a single QD-based sensor for sensitive measurement of DNA glycosylase activity at single-molecule level. The presence of DNA glycosylase may promote the removal of damaged DNA base from the biotin-labeled DNA substrate, generating an apurinic/apyrimidinic (AP) site. Subsequently the AP site is excised by apurinic/apyrimidinic endonuclease (APE1), leaving a single nucleotide gap. The Cy5-labeled dGTPs may be incorporated into the gapped sites with the assistance of DNA polymerase β, generating Cy5labeled DNA substrates. The resulting Cy5-labeled DNA substrates can be assembled onto the QD surfaces via specific biotin−streptavidin binding to form the QD−DNA substrate− Cy5 hybrids, inducing FRET from the QD to Cy5. Through simply monitoring the Cy5 signal by TIRF-based singlemolecule detection, the DNA glycosylase activity can be quantitatively determined. This single QD-based sensor exhibits excellent sensitivity with a detection limit of 1.8 × 10−6 U/μL without the involvement of any amplification, and it can be further applied for the enzyme kinetic analysis and the detection of endogenous DNA glycosylase activity in cell extracts.

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EXPERIMENTAL SECTION Chemicals and Materials. All oligonucleotides (Table 1) were synthesized by Takara Biotechnology Co. Ltd. (Dalian, Table 1. Sequences of the Oligonucleotidesa note

sequence (5′-3′)

sense strand antisense strand

CTC CTC CCC CAT CTC CTC CCA GTC C-biotin GGA CTG GGA GGA OAT GGG GGA GGA G

The underlined letter “O” in antisense strand symbolizes the damaged guanine (8-oxoG).

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China). Human 8-oxoguanine-DNA glycosylase 1 (hOGG1), 10× NEB buffer 2 (500 mM sodium chloride (NaCl), 100 mM trizma hydrochloride, 100 mM magnesium chloride (MgCl2), 10 mM DL-Dithiothreitol (DTT), 10 mg/mL bovine serum albumin (BSA), human apurinic/apyrimidinic endonuclease (APE1), pH 7.9), and 10× NEB buffer 4 (500 mM potassium acetate, 200 mM Tris-acetate, 100 mM magnesium acetate, 10 mM DTT, pH 7.9) were purchased from New England Biolabs (Ipswich, MA, U.S.A.). Thymine DNA Glycosylase (TDG) was bought from R&D System (Minneapolis, MN, U.S.A.). Human DNA polymerase β was purchased from Chimerx (Madison, WI, U.S.A.). Cyanine 5-dGTP (Cy5-labeled dGTP) was purchased from PerkinElmer (Foster City, CA, U.S.A.). Chromium(II) chloride (CdCl2), immunoglobulin G (IgG), and bovine serum albumin (BSA) were obtained from SigmaAldrich Company (St. Louis, MO, U.S.A.). The streptavidinconjugated 605QDs were obtained from Invitrogen Corpo-

Nt − No

Cy5 counting number when hOGG1 is absent, Nt is the Cy5 counting number when 0.1 U/μL hOGG1 is present, and Ni is the Cy5 counting number when 0.1 U/μL hOGG1 and CdCl2 are present. The IC50 value was calculated from the curve of RA versus the CdCl2 concentration. Cell Culture and Preparation of Cell Extracts. Human lung adenocarcinoma cell line (A549 cells) was cultured with B


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Analytical Chemistry Scheme 1. Principle of a Single Quantum Dot-Based Sensor for the hOGG1 Assaya

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The assay involves four steps: (1) hOGG1 removes the damaged 8-oxoG to generate an AP site; (2) the AP site is cleaved by APE1, leaving a single nucleotide gap; (3) the DNA polymerase β fills the gap by incorporating a Cy5-dGTP into the substrate, generating a Cy5-labeled substrate; and (4) the resulting Cy5-tagged DNA substrates may be coated onto the QD surface via biotin−streptavidin binding, inducing FRET from the QD to Cy5.

Figure 1. (A) Denaturating PAGE analysis of reaction products in the absence (left lane) and in the presence of hOGG1 (right lane), respectively. (B) Variance of QD and Cy5 fluorescence in the absence (control, blue line) and in the presence of hOGG1 (red line). The hOGG1 concentration is 0.1 U/μL.

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RESULTS AND DISCUSSION Principle of DNA Glycosylase Assay. As a proof of concept, we used the human 8-oxoguanine-DNA glycosylase 1 (hOGG1) as a model target. The hOGG1 participates in the repair of 8-hydroxyguanine (8-oxoG), which is a highly mutagenic oxidative DNA lesion in mammalian genome induced by reactive oxygen species (ROS).37 This assay involves the hOGG1-mediated fluorescent labeling and the subsequent QD-based FRET detection. The DNA substrate contains two complementary strands (i.e., sense strand and antisense strand). The sense strand is labeled with a biotin at the 3′ terminus, and the antisense strand is modified with an 8oxoG at the 13 nt site. The 8-oxoG residue may be specifically repaired through hOGG1-iniated base excision repair pathway.38 Specifically, hOGG1 first recognizes the 8-oxoG and cleaves the N-glycosidic bond between the sugar and the

10% fetal bovine serum (FBS) and 1% penicillin−streptomycin in Dulbecco’s modified Eagle’s medium. The cells were incubated at 37 °C in a humidified chamber with 5% CO2. The cells were collected in the exponential phase of growth with trypsinization and washed twice with ice-cold phosphatebuffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4), and pelleted at 1000 rpm at 4 °C for 5 min. The proteins were extracted by suspending in 100 μL of lysis buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (w/v) NP-40, 0.25 mM sodium deoxycholate, 1% (w/v) glycerol, and 0.1 mM 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride) and incubated on ice for 30 min and vortexed for 30 s every 5 min. After centrifugation at 12 000 rpm for 20 min at 4 °C, the supernatant was carefully transferred into a fresh tube and subjected immediately to hOGG1 activity assay. C


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Figure 2. Imaging of 605QD and Cy5 by TIRF. (A,B,C) hOGG1 is absent. (D,E,F) hOGG1 is present. The fluorescent signals of 605QD are shown in green (A,D) and the fluorescent signals of Cy5 are shown in red (B,E). The colocalization of 605QD and Cy5 is indicated by yellow color (F). The hOGG1 concentration is 0.1 U/μL. The scale bar is 2 μm.

TIRF imaging. The 605 nm-emitting QD (605QD) and Cy5 are chosen as the donor and the acceptor, respectively.39 Theoretically, the distance between the adjacent bases in dsDNA is about 0.34 nm.40 The theoretical distance between Cy5 and the 605QD in the 605QD-DNA substrate-Cy5 nanostructure is 12 nm, within the efficient FRET range (2R0 = 14 nm).39 As shown in Figure 1B, in the control experiment without hOGG1 (Figure 1B, blue line), no significant Cy5 signal is detected, indicating no FRET from the QD to Cy5, because no Cy5-labeled dGTP can be incorporated into the DNA substrates in the absence of hOGG1. On the contrary, the addition of hOGG1 induces the decrease of 605QD fluorescence intensity (Figure 1B, red line) and the increase of Cy5 fluorescence intensity. This result demonstrates the efficient FRET from the 605QD to Cy5 due to the formation of 605QD-DNA substrate-Cy5 nanostructure induced by hOGG1. Both the gel electrophoresis and fluorescence experiments (Figure 1) confirm that the hOGG1 may initiate the baseexcision-repair reaction in vitro and eventually lead to the generation of QD-DNA-Cy5 nanostructure. In addition, we measured the Cy5 signal at the excitation wavelength of 488 and 640 nm, respectively (see Supporting Information, Figure S2). In the control group without hOGG1, a distinct Cy5 signal can be observed at the excitation wavelength of 640 nm (direct excitation of Cy5), indicating the existence of Cy5 molecules; however, no Cy5 signal is observed at the excitation wavelength of 488 nm, suggesting no FRET between the 605QD and Cy5 due to the absence of 605QD-DNA-Cy5 nanostructure. In contrast, in the presence of hOGG1, Cy5 signals can be observed at the excitation wavelength of either 488 or 640 nm, indicating the efficient FRET between the 605QD and Cy5 due to the formation of 605QD-DNA-Cy5 nanostructure induced by hOGG1. Based on Figure 1B, the FRET efficiency is calculated to be F 82% according to E = 1 − FDA , where FD is the QD

damaged base, releasing the damaged base to form an apurinic/ apyrimidinic (AP) site. Then the AP endonuclease 1 (APE1) is introduced to cleave the AP site, leading to the generation of a single nucleotide gap. Subsequently, DNA polymerase β is employed to fill the gap by incorporating a Cy5-labeled dGTP at the gapped site, resulting in the formation of a Cy5-labeled DNA substrate (see Supporting Information, Figure S1). Because the DNA substrates are labeled with a biotin, the resulting Cy5-labeled DNA substrates may assemble onto the QD surface via specific biotin−streptavidin binding to form the QD-DNA-Cy5 hybrids, enabling FRET from the QD to Cy5. Consequently, the Cy5 signal may indicate the hOGG1 activity. Through simply monitoring the Cy5 signal by TIRF imaging, we may quantitatively measure the hOGG1 activity. In contrast, in the absence of hOGG1, the in vitro base excision repair reaction cannot be initiated, and thus, no Cy5-labeled dGTP can be incorporated into the DNA substrate, and no FRET signal is observed. Due to the high sensitivity of single-molecule detection and the high FRET efficiency of the QD-DNA-Cy5 nanostructure, the proposed method can be used for sensitive measurement of hOGG1 activity (Scheme 1). Validation of the Assay. This assay is dependent on the successful fluorescent labeling of DNA substrate by hOGG1mediated in vitro base excision repair reaction. To verify whether hOGG1 may initiate the first step of base excision repair (i.e., the cleavage of N-glycosidic bond between the sugar and the damaged base), we used denaturating gel electrophoresis to monitor the reaction products (Figure 1A). In the absence of hOGG1, only the 25 nt band (it contains both sense and antisense strand) is observed, indicating no occurrence of reaction. In contrast, after the incubation of hOGG1 with DNA substrates, the sense strand is cleaved, generating a product of 12 nt band. These results indicate that the hOGG1 can recognize and cleave the 8-oxoG-containg strand to initiate the base-excision-repair reaction. In living cells, dGTP may be further incorporated into the DNA substrates with the assistance of APE1 and DNA polymerase β. To verify whether the fluorophore-labeled dGTP may be incorporated into the DNA substrates by the in vitro base excision repair, we incubated the products with APE1 and DNA polymerase β, and subsequently, we mixed the resulting products with the QDs for

D

fluorescence intensity when hOGG1 is absent, and FDA is the QD fluorescence intensity when 0.1 U/μL hOGG1 is present. In theory, such a high FRET efficiency is reasonable. Because each 605QD is conjugated with 12−15 streptavidins and each streptavidin has 3 available biotin-binding sites after conD


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Analytical Chemistry jugation to the 605QD,39 up to 45 Cy5-labeled DNAs may be captured by a single QD. For a single QD with multiple acceptors, the FRET efficiency may be calculated on the basis of E =

nR 06 nR 06 + r 6

, where E is FRET efficiency, R0 is the Förster

distance, n is the average number of acceptor molecules interacting with one donor, and r is the average donor− acceptor separation distance. When the FRET efficiency is 82% and R0 is 6.9 nm (ref 39) and n is 45, the r is calculated to be 10.1 nm, which is close to the theoretical distance of 12 nm between Cy5 and the 605QD in the 605QD-DNA-Cy5 nanostructure. Taking into account the flexibility of DNA, the r value of 10.1 nm is reasonable. We further measured the fluorescence lifetime of 605QDs to confirm the efficient FRET between the 605QD and Cy5 in the 605QD-DNA-Cy5 nanostructure (see Supporting Information, Figures S3). The average lifetime of 605QD in the control group without hOGG1 is 15.1 ns. With the addition of 0.1 U/ μL hOGG1, the average lifetime of 605QD decreases to 3.5 ns due to the FRET between the 605QD and Cy5 as a result of the formation of 605QD-DNA-Cy5 nanostructure. The FRET τ efficiency is calculated to be 77% according to E = 1 − τDA ,

Figure 3. (A) Measurement of Cy5 counts generated by variableconcentration hOGG1. The inset shows the linear relationship between Cy5 counts and the hOGG1 concentration (logarithm) from 2 × 10−6 U/μL to 2 × 10−3 U/μL. Error bars represent standard deviations from three replicates.

= 136.1 + 23.6 log10 C (R2 = 0.9889), where N represents the Cy5 counts and C represents the hOGG1 concentration. We further calculated the detection limit to be 1.8 × 10−6 U/μL based on the principle of 3 times the standard deviation over the signal of the negative control. The sensitivity of the current assay has enhanced by 388.9-fold that of the AuNP-based colorimetric assay (7 × 10−4 U/μL),21 by 11.1-fold that of the luminescent assay (2 × 10−5 U/μL),42 and it is even comparable to that of the Exo III-assisted isothermal amplification-based fluorescent assay (1 × 10−6 U/μL).23 The enhanced sensitivity may result from (1) the efficient fluorescent labeling by in vitro base excision repair reaction, (2) the high FRET efficiency in the QD-DNA substrate-Cy5 nanostructure, and (3) the high sensitivity of single-molecule detection. Kinetic Analysis. We further employ the proposed method to evaluate the enzyme kinetic parameters of hOGG1. The initial velocities (V) are determined in the presence of hOGG1 (0.1 U/μL) and variable-concentration DNA substrate in 5 min reaction at 37 °C.43 The experimental data are fitted to the Michaelis−Menten equation V = Vmax[S]/(Km + [S]), where Vmax is the maximum initial velocity, [S] is the concentration of DNA substrate, and Km is the Michaelis−Menten constant. As shown in Figure 4, the maximum initial velocity (Vmax) is

D

where τDA is the lifetime of QD in the presence of hOGG1, τD is the lifetime of QD in the absence of hOGG1. This value is close to that obtained from Figure 1B by spectral measurement (82%). Measurement of hOGG1 Activity by TIRF Imaging. TIRF can only image the fluorescent molecules within 100 nm of the coverslip.41 Based on the separation distance of 12 nm between the 605QD and Cy5 in the 605QD-DNA-Cy5 nanostructure and the radius of 5.0−7.5 nm for streptavidinconjugated 605QD,39 we estimated that the largest distance between the fluorescent molecules (i.e., Cy5 and the 605QD) and the coverslip is 19.5 nm (much less than 100 nm), ensuring the efficient imaging of 605QD and Cy5 by TIRF. In the control group without hOGG1, only 605QD fluorescent signals are detected (Figure 2A), but no Cy5 signal is observed (Figure 2B). In contrast, when hOGG1 is present, both 605QD fluorescent signals (Figure 2D) and Cy5 fluorescent signals (Figure 2E) are simultaneously detected, with the yellow signals indicating the colocalization of 605QD and Cy5 (Figure 2F). In addition, the presence of hOGG1 induces the quenching of 605QD fluorescence due to FRET from the QD to Cy5 (Figure 2D), in agreement with the ensemble measurement (Figure 1B). These results clearly demonstrated that Cy5 signal can be used for the measurement of hOGG1 activity. Notably, when the FRET occurs in the presence of hOGG1, only the fluorescence brightness of QDs decreases (Figure 2D), but the number of visible QDs does not decrease obviously as compared with the control group without hOGG1 (Figure 2A). Taking into account the fact that the QDs are too bright to be completely quenched by FRET, we only used Cy5 count but not QD counts for hOGG1 assay. Detection Sensitivity. We measured the Cy5 signals in response to variable-concentration hOGG1 under the optimally experimental conditions (see Supporting Information, Figures S4−S7). When the hOGG1 concentration increases, the Cy5 counts significantly increase but reach a plateau above the concentration of 0.08 U/μL (Figure 3). Notably, there is a linear correlation between the Cy5 counts and the logarithm of hOGG1 concentration from 2 × 10−6 U/μL to 2 × 10−3 U/μL (inset of Figure 3). The corresponding equation is as follows: N

Figure 4. Variance of initial velocity (V) in response to various concentrations of DNA substrates. Error bars represent standard deviations from three replicates. E


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Analytical Chemistry determined to be 64.4 min−1 and Km is calculated to be 10.7 nM for hOGG1 in the presence of APE1. The Km value is consistent with that obtained by the standard radioactive assay (8.9 nM).43 These results suggest that the proposed method is suitable for kinetic analysis of hOGG1. Specificity of the Assay. We used several irrelevant proteins including bovine serum albumin (BSA), immunoglobulin G (IgG) and thymine DNA glycosylase (TDG) as the negative control samples to evaluate the detection specificity. BSA and IgG are not DNA glycosylases, and they cannot recognize the damaged bases. Even though TDG is a kind of DNA glycosylase, it can only selectively remove T from G/T mismatches.44 As shown in Figure 5, no significant Cy5 signal is

Figure 6. Variance of the relative activity of hOGG1 in response to variable-concentration CdCl2. The concentration of hOGG1 is 0.1 U/ μL. Error bars represent standard deviations from three replicates.

Figure 5. Measurement of Cy5 counts generated by the reaction buffer (control), 0.1 g/L BSA, 0.1 g/L IgG, 0.1 U/μL TDG, and 0.1 U/μL hOGG1. Error bars represent standard deviations from three replicates.

observed in the presence of reaction buffer (control), BSA, IgG, and TDG. In contrast, an extremely high Cy5 signal is detected when 0.1 U/μL hOGG1 is present. These results suggest that the proposed method possesses excellent specificity. Inhibition Assay. In this research, we used chromium(II) chloride (CdCl2, a classic inhibitor of OGG1) as a model inhibitor. The CdCl2 may inhibit hOGG1 activity through two approaches: (1) Cd2+ ion may bind to the enzyme molecules, leading to the inactivation of hOGG1; and (2) Cd2+ ions may bind to the enzyme−DNA substrate complex to prevent the cleavage of DNA substrates by OGG1.45 We measured the relative activity of hOGG1 in response to variable-concentration CdCl2, and we found that the relative activity of hOGG1 decreases when the CdCl2 concentration increases (Figure 6). The IC50 value is determined to be 10.93 μM, which is consistent with the IC50 value of 10 μM measured by radioactive assay.16 These results clearly demonstrate that the proposed method may provide a useful platform for the screening of hOGG1 inhibitors. Detection of Cellular hOGG1 Activity. The accurate detection of cellular hOGG1 activity is essential to biomedical research and clinical diagnosis. We use human lung adenocarcinoma cell line (A549 cells) as a model for the detection of cellular hOGG1 activity. Our result indicates that Cy5 counts increase when the number of A549 cells increases (Figure 7), with a linear correlation in the range from 5 to 10000 cells. The regression equation is N = 89.6 log10 X − 37.8 (R2 = 0.9905), where N represents the Cy5 counts and X represents the number of A549 cells, respectively. This result clearly demonstrates that this method can be applied for quantitative detection of hOGG1 activity in cell extracts.

Figure 7. Linear relationship between Cy5 counts and the number of A549 cells. Error bars represent standard deviations from three replicates.

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CONCLUSIONS In this research, we demonstrate the development of a single QD-based sensor for quantitative measurement of DNA glycosylase (hOGG1) activity. Taking advantage of the highly efficient in vitro base excision repair reaction, the enzyme activity information may be converted to a measurable fluorescence signal and subsequently imaged by TIRF. In comparison with the reported methods,15−19 the proposed method has a significant advantage of high sensitivity with a detection limit of 1.8 × 10−6 U/μL, which is much more sensitive than the reported colorimetric and luminescent assay.21,22,24 The proposed method may also be applied for the analysis of enzyme kinetic parameters and the screening of hOGG1 inhibitors. Because the DNA repair system is ubiquitous in nature,46 the proposed method may be extended to the detection of other DNA glycosylase and even other types of DNA repair enzymes by simply using the corresponding DNA repair pathways in vitro, thus offering a powerful tool for further applications in biomedical research and clinical diagnosis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b00664. F


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Additional information as noted in text (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86 0531-86180010. Fax: +86 0531-86180017. *E-mail: [email protected]. Tel.: +86 0531-86186033. Fax: +86 0531-82615258. Author Contributions †

These authors contributed equally (L.W. and F.M.).

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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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