Electrochemical Immunosensing Platform for DNA Methyltransferase

(2, 3) Normally, aberrant DNA methylation is always in contact with ... (18-22) Among them, electrochemical methods have attracted more and more atten...
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Electrochemical Immunosensing Platform for DNA Methyltransferase Activity Analysis and Inhibitor Screening Mo Wang, Zhenning Xu, Lijian Chen, Huanshun Yin,* and Shiyun Ai*

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College of Chemistry and Material Science, Shandong Agricultural University, Taian, 271018, PR China ABSTRACT: In this paper, we developed a novel electrochemical method to quantify DNA methyltransferase (MTase) and analyze DNA MTase activity. After the double DNA helix structure was assembled on the surface of gold nanoparticle modified glassy carbon electrode, it was first methylated by M. SssI MTase and then digested by restriction endonuclease HpaII, which could not recognize the methylated CpG site. Successively, anti-5-methylcytosine antibody was specifically conjugated on the CpG methylation site and horseradish peroxidase labeled goat antimouse IgG (HRP-IgG) was conjugated on anti-5-methylcytosine antibody. In the detection buffer solution containing H2O2 and hydroquinone, HRP-IgG can catalyze hydroquinone oxidation by H2O2 to generate benzoquinone, resulting in a highly electrochemical reduction signal. Consequently, the activity of M. SssI MTase was assayed, and DNA methylation was detected using the signal change with and without methylation. Furthermore, the inhibition investigation demonstrated that, in the presence of 160 μM S-adenosyl-L-methionine as methyl donor, 5-aza-2′-deoxycytidine, procaine, epicatechin, and caffeic acid could inhibit the M. SssI MTase activity with the IC50 values of 45.77, 410.3, 129.03, and 124.2 μM, respectively. Therefore, this study may provide a sensitive platform for screening DNA MTase inhibitors.

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using its oxidation signal. For example, Kato et al. quantitatively detected both C and mC bases of oligonucleotides by measuring the potential peak differences caused by C methylation with the electron cyclotron resonance nanocarbon film electrode.23 Wang et al. investigated the direct electrocatalytic oxidation of 5-mC in DNA without any bisulfite conversion, PCR amplification, or labeling procedures by multiwall nanotubes (MWNTs)/choline chloride/glassy carbon electrode (GCE).20 5-mC exhibited a significant electrochemical oxidation signal at 1.16 V in 0.1 M, pH 7.0 phosphate buffered saline (PBS). Because the oxidation potential of thymine is close to 5-mC, there was an interference in the detection of 5-mC in the presence of the thymine. Therefore, this strategy needed further improvement. The second strategy is to embed a molecule with electrochemical activity into the structure of hybridized DNA to recognize the methylated bases. For instances, Okamoto et al. designed a bipyridine-attached adenine derivative for sequence-specific osmium complexation, and this complexion could form a cross-link structure with hybridized DNA to distinguished methylated cytosines from unmethylated cytosines and was used to quantify the degree of methylation at a specific cytosine in the genome.24 However, though this method can specifically distinguish cytosine and 5mC, the synthesis of osmium complexation is more complicated, which might limit the application. The third strategy is based on the signal change of redox indicator before

NA methylation is a kind of epigenetics event that plays a crucial role in cell proliferation, gene transcription, and senescence.1 In eukaryotes, DNA methylation can only occur in the C-5 position of cytosine (C) with the region of CpG dinucleotides, and this process is catalyzed by specific DNA methyltransferases (MTase), using S-adenosyl-L-methionine (SAM) as the methyl donor.2,3 Normally, aberrant DNA methylation is always in contact with canceration because it can inactivate the tumor suppressor genes and lead to transcriptional silencing of gene expression.4,5 Actually, the overexpression of methylated DNA has been found in many cancers, such as thyroid tumors6 and human breast cancers.7 Therefore, development of simple, sensitive, and effective methods for DNA methylation detection and DNA MTase activity assay is important because it is in favor of early cancer diagnosis and understanding of the carcinogenesis mechanism. Up to now, several methods have been developed for the determination of DNA methylation and assay of MTases activity, such as methylation-specific polymerase chain reaction (PCR),8 real-time PCR-based methylation-specific PCR,9 colorimetry,10,11 direct microarray-based assay,12 fluorescence assay,13 single-molecule, real-time (SMRT) sequencing,14 HPLC,15,16 HPLC-MS,17 and electrochemical methods.18−22 Among them, electrochemical methods have attracted more and more attention for the DNA methylation assay in the last three years because of the advantages of cheap instruments, simple operation, easy separation, and high sensitivity and selectivity. The current electrochemical DNA methylation assay mainly contains three strategies. The first strategy is direct electrochemical determination of 5-methyl cytosine (5-mC) © 2012 American Chemical Society

Received: June 12, 2012 Accepted: October 2, 2012 Published: October 2, 2012 9072

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Scheme 1. Schematic Representation of the Developed Method for Detection of DNA Methylation and Assay of M. SssI MTase Activity

cytosine in DNA hybrid (5′-CCGG-3′) was methylated by MTase (M. SssI), anti-5-methylcytosine antibody was specially conjugated to 5-methylcytosine. Then, the anti-5-methylcytosine antibody was further specially conjugated to horseradish peroxidase labeled goat antimouse IgG (HRP-IgG). Consequently, HRP catalyzed the chemical oxidation of hydroquinone by H2O2 to form benzoquinone which resulted in a large electrochemical reduction signal of benzoquinone. Therefore, this reduction signal was used to assay the DNA MTase activity and detect DNA methylation. The HpaII endonuclease can recognize the duplex symmetrical sequence of 5′-CCGG-3′ and cleave the double-stranded DNA (dsDNA) between the two cytosines. After being digested by HpaII endonuclease, the dsDNA was cleaved at the M. SssI methylation sites and the CpG sequence could not be methylated. As a result, anti-5-methylcytosine antibody and HRP-IgG could not be immobilized on the electrode to catalyze the oxidation reaction and the electrochemical reduction signal would decrease. On the contrary, the cleavage effect of HpaII endonuclease could be hindered by the methylation of CpG sequence. Therefore, the electrochemical reduction signal after the cleavage of HpaII endonuclease was related to methylation level of the CpG sequence and the DNA MTase activity. On the basis of them, we realized the activity assay of M. SssI MTase and DNA methylation detection. In addition, the developed assay method can also be applied to screen the inhibitors of M. SssI MTase.

and after DNA (methylation or no methylation) is treated with DNA MTase and/or restriction enzyme. For instances, Liu et al. developed a novel method for electrochemical analysis of MTase activity and identification of DNA methylation. The hybridized DNA was methylated by M. SssI MTase and then cleaved by HpaII restriction enzyme. Then, the methylation status and MTase activity were evaluated from the voltammetric changes of electroactive indicator of ferroceneacetic acid, which was labeled at the end of probe DNA.21 Since DNA methylation plays an important role in prokaryotes and eukaryotes, the overexpression of DNA MTase in many cancer cells has been confirmed, and cytosine methylation of tumor suppressor genes in cancer cells seems to provide a highly specific target for pharmacologic therapy; the block of DNA methylation and/or the killing of the cancer cells through the inhibition of DNA MTase activity have attracted more and more attention. The research on DNA MTase inhibitors has been a hot field, and many potential inhibitors have been investigated. It has been reported that 5-aza-2′deoxycytidine has strong inhibitory effects on cancer cell growth in vitro and in vivo,25 and all the genes transcriptionally suppressed by DNA methylation in human hepatoma cells (HLE, HuH7, and HuH6 cells) were demethylated and reactivated with procaine treatment.26 It has also been proved that caffeic acid and chlorogenic acid (two common coffee polyphenols) can partially inhibit the methylation of the promoter region of the RARb gene in human breast cancer cells (MCF-7 and MAD-MB-231 cells).2 According to previous reports, one can conclude that the detection of DNA methylation, assay of MTase activities, and inhibitor screening have received more and more research interest. It has been reported that anti-5-methylcytosine antibody can specifically cross-react with methylated cytosine in both single-stranded and double-stranded DNA.27 On the basis of this, this work reported an electrochemically immune approach for the assay of MTase activity and the detection of DNA methylation of specific CpG sites (Scheme 1). After the

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EXPERIMENTAL SECTION

Reagents and Materials. Hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O), procaine hydrochloride, tris(hydroxymethyl)aminomethane (Tris), tris(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), epicatechin, and caffeic acid were purchased from Aladdin (Shanghai, China). Polyethylene glycol 3350 (PEG-3350) was purchased from Solarbio (Beijing, China). 5-Aza-2′-deoxycytidine and catecholO-methyltransferase (COMT) were purchased from Sigma9073

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Aldrich (St. Louis, USA). M. SssI MTase and restriction endonuclease HpaII were supplied by New England BioLabs (Ipswich, MA) and Fermentas (Maryland, USA), respectively. According to the supplier, the methyltransferase M.SssI was isolated from a strain of Escherichia coli transfected with the M.SssI methyltransferase gene from the Spiroplasma sp. strain MQ1, and it is stored at −20 °C in a buffer containing 10 mM Tris−HCl (pH 7.4), 50 mM NaCl, 1 mM dithiothreitol, and 10 mM MgCl2. Restriction endonuclease HpaII was isolated from Haemophilus parainf luenzae, and it was stored at −20 °C in a buffer containing 10 mM Tris−HCl (pH 7.4), 100 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol, 0.2 mg/mL bovine serum albumin (BSA), and 50% glycerol. Anti-5-methylcytosine antibody was purchased from Calbiochem (Merck KGaA, Darmstadt, GER). Ten μg/mL anti-5-methylcytosine antibody was prepared with 0.1 M PBS (pH 7.4). HRP-IgG and the synthetic oligonucleotides were purchased from Shanghai Sangon Biotechnology Co. (Shanghai, China) and used without further purification. HRP-IgG was diluted 100 times with 10 mM phosphate buffer solution (PBS, pH 7.0). The base sequences of oligonucleotides are as follows: thiol-capped probe DNA (DNA S1), 5′-SH-(CH2)6-TAG TGT GAT GTC ACC TAG TTG ACC TTC CGG AT-3′; target DNA (DNA S2), 5′-ATC CGG AAG GTC AAC TAG GTG ACA TCA CAC TA-3′; one-base mismatched DNA (DNA S3), 5′-ATC CTG AAG GTC AAC TAG GTG ACA TCA CAC TA-3′. The bold font of CCGG was the recognized bases by M. SssI methyltransferase and HpaII restriction endonuclease. The bold/italic font of T was the mismatched base. The synthesized oligonucleotides were diluted in TE buffer (containing 10 mM Tris-HCl and 1 mM EDTA, pH 8.0) to desired stock concentrations and stored at −20 °C according to the manufacturer’s instructions. The buffer solutions employed in this study are as follows. Probe immobilization buffer: 10 mM Tris-HCl, 1.0 mM EDTA, 1.0 M NaCl, and 1.0 mM TCEP (pH 7.0); DNA hybridization buffer: 10 mM Tris-HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.4); electrochemistry determination buffer: 0.1 M phosphate buffered saline (PBS, pH 7.0). All reagents were analytically pure grade. All of the solutions and redistilled deionized water used were autoclaved. Electrochemical experiments were performed at CHI660C electrochemical workstation (USA) with a conventional threeelectrode cell. A bare GCE or modified GCE was used as working electrode. A saturated calomel electrode (SCE) and a platinum wire were used as the reference electrode and auxiliary electrode, respectively. Probe Immobilization and Hybridization. The GCE was polished with 0.03 μm alumina powder on a microcloth pad and rinsed thoroughly with redistilled deionized water. Then, the polished electrode was washed successively with redistilled deionized water, anhydrous ethanol, and redistilled deionized water in an ultrasonic bath. After being dried with nitrogen blowing, the electrode was immersed into 3 mM HAuCl4 solution containing 0.1 M KNO3 and the gold nanoparticles (AuNPs) were electrochemically deposited on the electrode surface by single-potential mode using the amperometry technique at −0.2 V for 300 s. Finally, the electrode was washed with redistilled deionized water and dried at room temperature. The obtained electrode was noted as AuNPs/GCE. For probe immobilization, 5 μL of probe immobilization buffer containing 5.0 × 10−7 M thiol-capped probe DNA S1

was dripped on AuNPs/GCE surface and maintained for 1.5 h at humid conditions. Then, the probe modified electrode was rinsed three times with probe immobilization buffer for 15 min. After that, 5 μL of Tris-HCl (10 mM) containing 1.65 × 10−6 mM MCH was dripped on the electrode surface and kept for 1 h to hold a good orientation of probe DNA for its good recognition ability. The hybridization experiments were carried out by dripping 5 μL of DNA hybridization buffer containing 5.0 × 10−7 M of target DNA S2 on the electrode surface at 37 °C for 2 h. After that, the hybridized electrode was rinsed three times with hybridization buffer to remove the unhybridized target DNA and dried with nitrogen blowing. Methylation and Assay of M. SssI MTase Activity. The methylation S1/S2 hybrid was performed at 37 °C for 2 h in 10 mM Tris-HCl buffer (pH 7.0) containing 160 μM SAM, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), and various concentrations of M. SssI (from 0 to 80 unit/mL). Anti-5-methylcytosine Antibody and HRP-IgG Immobilization. After methylation, the electrode was incubated in 0.05% PEG-3350 solution (prepared in 0.1 M PBS) for 30 min to prevent any possible nonspecific binding. After the electrode was rinsed three times with 0.1 M PBS (pH 7.0) for 15 min, 5 μL of anti-5-methylcytosine antibody (10 μg/mL) was dripped on the electrode surface and incubated for 1 h. Then, the electrode was rinsed with 0.1 M PBS for 15 min. Subsequently, 5 μL of HRP-IgG (1:100) was dripped on the electrode surface and incubated for 30 min at room temperature. The electrode was then rinsed three times with 0.01 M PBS and dried at room temperature. Cleavage of HpaII Endonuclease. HpaII digestion was performed at 37 °C in 10 mM Tris-HCl buffer (pH 7.4) containing 50 unit/mL HpaII, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 200 μg/mL bovine serum albumin (BSA), and 50% glycerol for 2 h. After digestion, the electrode was thoroughly washed with 10 mM Tris-HCl (pH 7.4) and further treated as described in Anti-5-methylcytosine Antibody and HRP-IgG Immobilization. Then, the electrode was transferred into 0.1 M PBS (pH 7.0) to record the electrochemical response. Inhibition of Dam MTase Activity. To study the inhibition effects of several compounds, 5-aza-2′-deoxycytidine, procaine, epicatechin, and caffeic acid on the M. SssI activity, the methylation of S1/S2 hybrid was performed at 37 °C in 10 mM Tris-HCl buffer (pH 7.0) containing 160 μM SAM, 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 50 unit/mL M. SssI, and various concentrations of the inhibitors. For caffeic acid and epicatechin, COMT was also added into the inhibitory system. The inhibition efficiency (%) was estimated as follows: Inhibition (%) =

I2 − I3 × 100% I2 − I1

where I1 was the peak current of the H2O2−hydroquinone system obtained after the S1/S2 hybrids successively treated with M. SssI, HpaII, and anti-5-methylcytosine antibody, I2 was the peak current of the H2O2−hydroquinone system obtained after the S1/S2 hybrids successively treated with M. SssI, HpaII, anti-5-methylcytosine antibody, and HRP-IgG, and I3 was the inhibited peak current of the H2O2−hydroquinone system. Electrochemical Determination. Differential pulse voltammetry (DPV) was performed in 10 mL of 0.1 M PBS (pH 7.0) containing 50 μM H2O2 and 50 μM hydroquinone. The 9074

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than that at Figure 1, curve e, indicating that the digestion of S1/S2 hybrids by HpaII endonuclease was blocked by CpG methylation. We also evaluated the methylation specificity by hybridizing the probe DNA S1 with one-base mismatched DNA S3. After immobilization of probe DNA S1, it was hybridized with DNA S3 and then treated successively with M. SssI, anti-5methylcytosine antibody, and HRP-IgG. It was obvious that the reduction signal of benzoquinone (Figure 1, curve g) was lower than the signal after probe DNA S1 hybridized completely with complementary DNA S2, indicating that the hybrid of S1 and S3 can not be recognized by M. SssI MTase because the hybrid does not contain a specific recognition sequence (5′-C/CGG-3′). This result also proved that the developed method could discriminate even one-base mismatched DNA when the mismatched base was located at the recognition site of M. SssI MTase. Moreover, this method can also be used to selectively detect CpG DNA methylation. The repeatability of the developed method was evaluated by recording the reduction current of benzoquinone in 0.1 M PBS containing 50 μM H2O2 and 50 μM hydroquinone after the hybrids were treated successively with 50 unit/mL M. SssI MTase, 10 μg/mL anti-5-methylcytosine antibody, and HRPIgG (1:100). The RSD (relative standard deviation) for six independent treatment hybrids was 6.77%, indicating an acceptable repeatability of the developed method. However, it must be addressed that the DNA methylation detection using this method was dependent on the location of the methylation and the application ability of our method would be limited by DNA concentration because PCR polymerases cannot amplify or incorporate methylated cytosines into their amplicon products. Assay of M.SssI MTase Activity. The catalytic activity and availability of M. SssI were investigated by changing the M. SssI concentration from 0.05 to 80 unit/mL. The assay process was as follows. After the hybridization of DNA S1 and DNA S2 at the electrode surface, the hybrids were successively treated with different concentrations of M. SssI (from 0.05 to 80 unit/mL) and 50 unit/mL HpaII for 2 h, respectively. After that, the hybrids were further successively treated with 10 μg/mL anti-5methylcytosine antibody and HRP-IgG (1:100) for 1 h and 30 min, respectively. Finally, the DPV response of benzoquinone was recorded in 0.1 M PBS containing 50 μM H2O2 and 50 μM hydroquinone. The reduction current increased with increasing the M. SssI concentration (Figure 2A), which can be ascribed to the fact that more M. SssI MTase can methylate more cytosine in CpG island. The symmetrical methylated cytosine in S1/S2 hybrids can capture more anti-5-methylcytosine antibody and HRP-IgG on the electrode surface. As a result, the reduction current increased gradually. As seen in Figure 2B, the reduction current increased linearly with the M. SssI concentration from 0.5 to 50 unit/mL and followed the regression equation of I = 0.0339c + 1.914 (μA, unit/mL, R = 0.9971). The detection limit was 0.1 unit/mL (S/N = 3). When the concentration of M. SssI was higher than 50 unit/mL, the reduction current trended to level off, which indicated that almost all the S1/S2 hybrids were methylated and avoided the cleavage by HpaII. As a consequence, the concentration of benzoquinone achieved a stable value because no more anti-5-methylcytosine antibody and HRP-IgG were conjugated to the hybrids to further catalyze the oxidation reaction. The detection precision was investigated according to the slope of the regression equation of M. SssI (from 0.5 to 50 unit/mL) obtained from three

parameters were as follows: increment potential, 0.004 V; pulse amplitude, 0.05 V; pulse width, 0.05 s; sample width, 0.0167 s; pulse period, 0.2 s; quiet time, 2 s.

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RESULTS AND DISCUSSION DNA Methylation Detection. As shown in Figure 1, a well-defined reduction peak at about 0.035 V was observed in

Figure 1. Differential pulse voltammograms of the biosensor in 0.1 M PBS (pH 7.0) after different treatments. (a) Probe DNA S1 modified AuNPs/Au. (b) Probe DNA S1 hybridized with complementary DNA S2. (c) The S1/S2 hybrids were treated successively with M. SssI MTase and anti-5-methylcytosine antibody. (d) The S1/S2 hybrids were treated successively with M. SssI MTase, anti-5-methylcytosine antibody, and HRP-IgG. (e) The S1/S2 hybrids were first digested with HpaII restriction endonuclease and then treated the same as curve d. (f) The S1/S2 hybrids were first treated with M. SssI MTase, then digested with HpaII restriction endonuclease, and finally treated successively with anti-5-methylcytosine antibody and HRP-IgG. (g) Probe DNA S1 was hybridized with single-base mismatched DNA S3.

H2O2−hydroquinone solution after probe DNA S1 was immobilized on the AuNPs/Au electrode surface (Figure 1, curve a). This signal can be ascribed to the reduction of benzoquinone originated from the oxidation of hydroquinone by H2O2. However, the reduction peak current decreased successively after the probe DNA S1 hybridized with DNA S2 (Figure 1, curve b) and the hybrids were further treated with M.SssI MTase and anti-5-methylcytosine antibody (Figure 1, curve c). After the double-stranded DNA (S1/S2) was treated successively with M.SssI MTase, anti-5-methylcytosine antibody, and HRP-IgG, the electrochemical reduction signal increased significantly due to the catalytic activity of HRP (Figure 1, curve d). This result also demonstrated that the hybrids of DNA S1 and DNA S2 had been successively methylated by M. SssI MTase, and the anti-5-methylcytosine antibody and HRP-IgG had been specifically conjugated on the methylation sites. However, when the S1/S2 was first digested by HpaII endonuclease at the recognized sites of the site of 5′C/CGG-3′ and then treated successively with CpG MTase (M. SssI), anti-5-methylcytosine antibody, and HRP-IgG, the electrochemical reduction signal decreased significantly because the S1/S2 could not be methylated and HRP could not be immobilized on the electrode surface to catalyze the oxidation reaction (Figure 1, curve e). To further prove the methylation event, the hybrids of S1 and S2 were incubated in M. SssI MTase solution, then digested with HpaII endonuclease, and finally treated with anti-5-methylcytosine antibody and HRPIgG. As seen in Figure 1, curve f, the reduction signal of benzoquinone was the same in Figure 1, curve d, and higher 9075

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Figure 2. (A) DPV response of the biosensor treated with different concentrations of M. SssI MTase. (B) The DPV response current obtained with different concentration of M. SssI MTase from 0.05 to 80 unit/mL. (C) DPV response of the biosensor treated with 50 unit/ mL M. SssI MTase with different times. (D) The DPV response current obtained with different treatment times from 0 to 160 min.

Figure 3. The inhibition effect of 5-aza-2′-deoxycytidine (A), procaine (B), epicatechin (C), and caffeic acid (D) on M. SssI MTase activity.

implied that the inhibition of 5-aza-2′-deoxycytidine was in a dose-dependent manner, which was in accord with a previous report.25 It had also been reported that the inhibitor mechanism for 5-aza-2′-deoxycytidine was a competition process; that is, in the presence of 5-aza-2′-deoxycytidine, the donative methyl from SAM could be simultaneously transferred to 5-aza-2′-deoxycytidine and cytosine residue in DNA under the catalysis of DNA MTase. On the basis of this, the methylation level of cytosine residue in DNA decreased gradually along with the increased concentration of inhibitor. As a consequence, the conjugate amount of anti-5-methylcytosine antibody and HRP-IgG (1:100) reduced gradually and the reduction current decreased. The maximum inhibition of 5aza-2′-deoxycytidine was 75.15% with the IC50 value of 45.77 μM. Procaine, a kind of local anesthetic, is a potential DNAdemethylating agent with growth-inhibitory effect in a human breast cancer cell (MCF-7 cells), human hepatoma, and hepatoblastoma cell lines (HLE, HuH7, and HuH6 cells).26,33 Procaine can cause global genomic DNA hypomethylation and demethylation and reactivation of tumor suppressor genes with hypermethylated CpG islands.33 Therefore, the inhibition effect of procaine was also investigated in this work (Figure 3B). Same as 5-aza-2′-deoxycytidine, the reduction current first decreased gradually with increasing the concentration, indicating that procaine worked as DNA methylation inhibitor. Then, the reduction currents trended to level off when the inhibitor concentrations achieved a certain level. These results were in accord with that procaine could strongly bind to CpG DNA to block the methylation event.33 The maximum inhibition ratio was 49.09% with the IC50 value of 410.3 μM. It is known that various polyphenols containing catechol structure are excellent substrates for the COMT-mediated Omethylation with SAM as methyl donor.34,35 The COMTmediated rapid methylation of catechols would not only significantly consume SAM but also form equimolar amounts of S-adenosyl-L-homocysteine (SAH), which is the demethylated SAM and a feedback inhibitor of various SAM-dependent methylation processes.36,37 Therefore, in this work, we investigated not only the inhibition efficiency of two polyohenols (epicatechin and caffeic acid) but also the effect of COMT on DNA methylation inhibition. When COMT was

independent assay processes. The RSD of the three slopes was 4.38%. This result demonstrated that the developed assay method had good precision. The effect of methylation time on the methylation level catalyzed by M. SssI was also investigated by DPV. As seen in Figure 2C,D, the reduction current of benzoquinone increased with extending the methylation time from 0 to 120 min, which was in accordance with the fact that more amount of cytosine in CpG island could be methylated by the long treatment time of M. SssI MTase. Then, the reduction current increase gradually became slower, caused by the consumption of substrates. Finally, the reduction current leveled off. This phenomenon may be explained by the saturated methylation of cytosine in CpG island. Assay of the Inhibition of M. SssI MTase Activity by Different Inhibitors. Aberrant DNA methylation is always related to disease, especially cancer;6,7 therefore, the screening of DNA MTase inhibitors plays an important role in disease treatment and receives more and more attention. A kind of excellent DNA MTase inhibitor can effectively block the transfer of the methyl group in SAM to the basic group in DNA; the specific sites for methylation can not be recognized, and the reduction signal of benzoquinone will decrease compared with the control. To date, the reported small molecule DNA MTase inhibitors mainly contain two categories, nucleoside analogs (such as 5azacytidine,28 5-aza-2′-deoxycytidine,29 Zebularine,30 and 5fluoro-2′-deoxycytidine26) and non-nucleoside analogs (such as procaine,26 fisetin, myricetin, quercetin, caffeic acid, catechin, and epicatechin31). In order to test and verify that the developed method can be employed for the screening of M. SssI MTase inhibitors, four kinds of compounds were selected as model inhibitors, such as 5-aza-2′-deoxycytidine, procaine, epicatechin, and caffeic acid, which had been proved with the inhibition activity toward DNA methylation.2,25,29,31,32 For 5-aza-2′-deoxycytidine (Figure 3A), the reduction current first decreased gradually with increasing the concentration. Then, the reduction currents trended to level off when the inhibitor concentrations achieved a certain level. This result 9076

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not present in the inhibition system, these polyphenols showed weak inhibitory efficiency on the DNA methylation mediated by M. SssI MTase (Figure 3C,D). However, in the presence of COMT, the inhibitory efficiency improved significantly and the inhibitions were also dose dependent as 5-aza-2′-deoxycytidine and procaine. Under the selected experimental conditions, the maximum inhibition efficiencies were 69.69 and 66.67% with the IC50 values of 129.03 and 1124.2 μM for epicatechin and caffeic acid, respectively. On the basis of these results, one can conclude that the developed method can be used to screen the inhibitors toward DNA methylation. It seems that these IC50 values are a little high, which might be caused by the high concentration of SAM (160 μM) because the IC50 value were dose dependent on SAM. That is, if the concentration of SAM was high in the inhibition system, the IC50 value might be high; if the concentration of SAM was low in the inhibition system, the IC50 value might be low. In fact, the concentration of SAM can not achieve a level of 160 μM in humans; therefore, the dosage required for therapy does not need to be at the concentration of IC50 values obtained in our work. From Figure 3, it can also be concluded that the four inhibitors have different activity on blocking DNA methylation. The maximum inhibition efficiency of 5-aza-2′-deoxycytidine was about 75.15%, which was much higher than that at procaine (49.09%), caffeic acid (66.67%),and epicatechin (69.69%). Moreover, it must be highlighted that the concentrations of quercetin, caffeic acid, and epicatechin were much higher than 5-aza-2′-deoxycytidine when the maximum inhibition efficiency was obtained, which indicated that 5-aza2′-deoxycytidine was the most effective inhibitor for M. SssI MTase. These results proved that the developed method was suitable to investigate the inhibitory effects of anticancer drugs on DNA MTase and screening DNA MTase inhibitors.

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CONCLUSION In summary, we developed a novel electrochemical method for detection of DNA methylation, assay of DNA MTase activity, and screening of inhibitors. By combining the discriminative capacity of M. SssI MTase, specific conjugation capacity of anti5-methylcytosine antibody, and catalytic ability of HRP-IgG, the fabricated biosensor can easily distinguish a DNA methylation event with 5.0 × 10−7 M DNA. Compared with the traditional method, this method does not need sophisticated and expensive instrument, and a low detection limit was obtained, 0.1 unit/mL. In addition, our developed method could also be applied to analyze the activity of other DNA MTases and screen their inhibitors using the corresponding DNA sequence.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 538 8247660. Fax: +86 538 8242251. E-mail: yinhs@ sdau.edu.cn (H.Y.); [email protected] (S.A.). 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 (Nos. 21075078, 21105056) and the Natural Science Foundation of Shandong province, China (Nos. ZR2010BM005, ZR2011BQ001). 9077

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