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DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity
Analytica Chimica Acta 829 (2014) 9–14
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DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity Zhenning Xu, Huanshun Yin, Yunxiang Han, Yunlei Zhou *, Shiyun Ai * College of Chemistry and Material Science, Shandong Agricultural University, 61 Daizong street, Taian, Shandong 271018, PR China
H I G H L I G H T S
A new electrochemical protocol was fabricated. The DNA-based hybridization chain reaction was used for signal amplification. Fluorouracil and daunorubicin hydrochloride could inhibit the DNA MTase activity. Bisphenol A and nonyl phenol could improve the DNA MTase activity.
G R A P H I C A L A B S T R A C T
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A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2013 Received in revised form 9 April 2014 Accepted 11 April 2014 Available online 24 April 2014
In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 unit mL1. Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M. SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism. ã 2014 Elsevier B.V. All rights reserved.
Keywords: DNA methylation Hybridization chain reaction Methyltransferase Inhibitors Environmental phenolic hormones
1. Introduction
* Corresponding authors. Tel.: +86 538 8249248/7660; fax: +86 538 8242251. E-mail addresses: [email protected] (Y. Zhou), [email protected], [email protected] (S. Ai). http://dx.doi.org/10.1016/j.aca.2014.04.024 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
DNA methylation is the principal driving forces behind the phenomenon of epigenetic [1]. DNA methylation could modulate gene expression and genomic integrity [2], which was catalyzed by methyltransferases (MTase) in the presence of S-adenosyl-L-
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methionine (SAM) as methyl donor. It has been proposed that the ancestral function of DNA methylation could actually restrain the spread of parasitic elements. Aberrant DNA methylation was the most common molecular lesion of the cancer cell [3]. Moreover, aberrant DNA methylation has also been found to be related to the aberrant DNA MTase activity [4,5]. In this regard, we expect to develop a novel sensitive and selective method to detect DNA methylation and DNA MTase activity. Traditional analytical methods such as bisulfites methods [6,7], HPLC (high performance liquid chromatography) [8], fluorescence methods [9,10] and PCR (polymerase chain reaction)-based techniques [11] have been widely used for the determination of DNA methylation. These methods, however, required complicated pretreatment steps, extensive labor resources, and are not applicable for simple and sensitive determination. As known, electrochemical method is the most popular method for DNA methylation and DNA MTase activity with sample preconcentration, time-saving, high sensitivity and selectivity [4,12–15]. Meng et al. has distinguished cytosine and 5-methylcytosine (5-mC) at low potentials by sodium dodecyl sulfate functionalized graphene modified pyrolytic graphite electrode [16]. However, there was an interference caused by thymine (T) because of the almost same oxidation potentials of T and 5-mC [15]. Therefore, direct determination of 5-mC needed further improvement. The DNA MTase and/or restriction enzyme was used in many reports about DNA methylation detection [4,13,17,18]. However, the detection sensitivity needs to be further improved. In 2004, Dirks and Pierce introduced the concept of hybridization chain reaction (HCR). The stable DNA monomers assembled only upon exposure to a target DNA fragment [19]. Tang’s group reported a novel electrochemical immunoassay for the determination of proteins at an ultralow concentration using DNA-based HCR [20]. Chen et al. proposed a strategy based on HCR signal amplification to detect the sequence specific DNA [21]. Compared with traditional construction, HCR could effectively amplify the signal and gain higher sensitivity [22– 24]. Herein, we described an electrochemical method for the detection of DNA methylation and the assay of the
methyltransferase activity based on HCR signal amplification (Scheme 1). After the dsDNA digested by HpaII, the DNA hybrid was cleaved at a specific site 50 -CCGG-30 and the nucleotide sticky end in the target DNA disappeared. However, the cleavage of the HpaII endonuclease was blocked by CpG methylation. When the symmetrical cytosine residues in dsDNA was methylated by M. SssI MTase and digested with HpaII restriction endonuclease, the HCR of DNA initiator strands reacted between biotinylated H1 and H2 hairpin DNA molecules. The streptavidin-alkaline phosphatase (S-ALP) could be conjugated on the electrode surface through the specific interaction between biotin and S-ALP. The ALP molecules could catalytically hydrolyze 1-naphthyl phosphate to produce 1naphthol for generating amplified electrochemical signal. The electrochemical signal was used to quantify the M.SssI MTase activity and screen the inhibitors of M.SssI MTase. 2. Materials and methods 2.1. Reagents Tris(hydroxymethyl) aminomethane (Tris), Tri(2-carboxyethyl) phosphine hydrochloride (TCEP), hydrogen tetrachloroaurate trihydrate (HAuCl43H2O), mercaptopropronic acid (MPA) was obtained from Alfa Aesar (Heysham, Lancashire, UK). Disodium ethylenediaminetetraacetic acid (EDTA), 5-fluorouracil, bisphenol A and nonylphenol were purchased from Aladdin (Shanghai, China). Daunorubicin HCl was purchased from Di Bo Chemical Technology Co., Ltd. (Shanghai, China). S-ALP and PAGE-purified DNA were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The base sequences are as follows: probe DNA (DNA S1): 50 SH-TAG TGT GAT GTC ACC TAG TTG ACC TTC CGG AT-30 , target DNA (DNA S2): 50 -GAA GGA GGG GCG ACT ATC CGG AAG GTC AAC TAG GTG ACA TCA CAC TA-30 , one-base mismatched DNA (DNA S3): 50 GAA GGA GGG GCG ACT ATC CTG AAG GTC AAC TAG GTG ACA TCA CAC TA-30 , hairpin probe H1: (DNA S4): 50 -GGG GCG ACT TGA AAC AGT CGC CCC TCC TTC-biotin-30 , hairpin probe H2: (DNA S5): 50 biotin-GTT TCA AGT CGC CCC GAA GGA GGG GCG ACT-30 . The recognition sites of 50 -CCGG-30 for M.SssI MTase and HpaII
Scheme 1. Schematic illustration of electrochemical method for detection of DNA methylation and assay of DNA MTase activity.
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endonuclease were marked with bold font. The mismatched base T was marked with bold/italic. The synthesized oligonucleotides were diluted in TE buffer to desired stock concentrations and stored at 20 C. M.SssI MTase and restriction endonuclease HpaII are supplied by New England BioLabs (Ipswich, MA) and Fermentas (Maryland, USA), respectively. They were stored at 20 C in a 50 mM Tris–HCl buffer (pH 7.5) containing 10 mM EDTA, 50 mM KCl, 200 mg mL1 bovine serum albumin (BSA), 25% glycerol, 1 mM dithiothreitol (DTT). The buffer solutions for electrochemistry were as follows: Oligonucleotide dissolve buffer (TE buffer): 10 mM Tris–HCl and 1 mM EDTA (pH 8.00); probe immobilization buffer: 10 mM Tris– HCl, 1.0 mM EDTA, 1.0 M NaCl and 1.0 mM TCEP (pH 7.00); DNA hybridization buffer: 10 mM Tris–HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.00); electrochemistry determination buffer: 0.1 M Tris–HCl and 1 mM Mg(NO3)2 (pH 9.80). The pH of buffer solutions was adjusted by adding 0.1 M HCl or 0.1 M NaOH. All reagents were analytically pure grade. All of the solutions and redistilled deionized water were autoclaved. 2.2. Gold nanoparticle deposition A gold electrode was polished carefully to a mirror-like surface with 0.3 mm and 0.05 mm alumina slurry and then sequentially sonicated for 3 min in absolute ethanol and double distilled deionized water, respectively. Prior to the experiment, the bare gold electrode was cyclic-potential scanned within the potential range of 0.2–1.8 V in 0.2 M H2SO4 until a voltammogram characteristic of the clean polycrystalline gold was established. Gold nanoparticle was deposited onto bare gold electrode in an aqueous electrolyte of 3 mM HAuCl4 solution containing 0.1 M KNO3 at a constant potential of 0.2 V for 200 s under stirring. The gold electrodes modified with gold nanoparticles were rinsed with water and dried with N2 for further experiments. The obtained electrode was noted as AuNPs/Au. 2.3. Probe immobilization and hybridization For assembly of probe on AuNPs/Au surface, 5 mL of 0.5 mM thiol-capped probe DNA S1 in probe immobilization buffer was dropped on AuNPs/Au surface and maintained for 12 h at humid conditions. The electrode was thoroughly washed with 10 mM Tris–HCl (pH 7.40) for three times. Then 5 mL Tris–HCl (10 mM, pH 7.40) containing 3 mM MCH was dropped on the electrode surface and kept for 1 h to eliminate the un-immobilized probe DNA S1 for its good recognition ability. Thus, the ssDNA/AuNPs/Au was obtained. The hybridization experiment was performed by dripping 5 mL DNA hybridization buffer containing 0.5 mM of target DNA S2 on the electrode surface and the hybridization process was kept at 37 C for 2 h. After that, the electrode was rinsed three times with 10 mM Tris–HCl (pH 7.40) to remove the un-hybridized target DNA and dried with nitrogen blowing. The obtained electrode was named as dsDNA/AuNPs/Au. 2.4. Methylation and cleavage For methylation process, the dsDNA was treated with 10 mM Tris–HCl buffer (pH 7.50) containing 160 mM SAM, 50 mM KCl, 10 mM EDTA, 200 mg mL1 BSA, 25% glycerol, 1 mM DTT and various concentrations of M.SssI (from 0 to 80 unit mL1 at 37 C for 2 h. Then, the obtained electrode was rinsed with 10 mM Tris–HCl (pH 7.40) for three times. And then, the above electrode was digested by HpaII endonucleases at 37 C in 10 mM Tris–HCl buffer (pH 7.50) containing 50 unit mL1 HpaII, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, and 50% glycerol for 2 h. After
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digestion, the electrode was thoroughly washed with 10 mM Tris– HCl (pH 7.40). 2.5. HCR and S-ALP tagging After washing with Tris–HCl, the modified electrode was incubated with 5 mL hybridization buffer containing biotinylated hairpin probe H1 (2.5 mM) and H2 (2.5 mM) for 3 h. The electrode was thoroughly washed with 10 mM Tris–HCl (pH 7.40) and the obtained electrode was named as (H2/H1)n/dsDNA/AuNPs/Au. After that, 5 mL S-ALP was soaked on the electrode surface for 30 min at room temperature. The electrode was then rinsed three times with 10 mM Tris–HCl buffer (pH 7.40) and dried at room temperature and was noted as S-ALP/(H2/H1)n/dsDNA/AuNPs/Au. 2.6. Inhibition the activity of M.SssI MTase To further study the activity of M.SssI, 5-fluorouracil, daunorubicin HCl, nonylphenol and bisphenol A were used to the model inhibitors. The DNA S1/S2 hybrids were performed at 37 C for 2 h in 10 mM Tris–HCl (pH 7.40) containing 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, 50% glycerol, 160 mM SAM, 50 unit mL1 M.SssI and various concentration of inhibitors. The inhibition efficiency (%) was estimated as follows: Inhibitionð%Þ ¼
I I1 I
where I is the peak current of 1-naphthol obtained after the S1/S2 hybrids successively treated with M.SssI, HpaII, H1, H2 and S-ALP. I1 is the inhibited peak current of 1-naphthol. 2.7. Electrochemical determination The electrode was dipped into a stirring Tris–HCl containing 1 mM Mg(NO3)2 (pH 9.80) and 1.0 mM 1-naphthyl phosphate for 3 min. Differential pulse voltammetry (DPV) was performed with a CHI 660C electrochemical workstation (Austin, USA). The measurements were based on a conventional three-electrode system. The parameters are 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, 6 s. Electrochemical impedance spectroscopy (EIS) was performed with a CHI 660C electrochemical workstation in 5 mM [Fe(CN)6]3/4 (1:1) solution containing 0.1 M KCl as the supporting electrolyte. The frequency was ranged from 101 to 105 Hz. 3. Results and discussion 3.1. Characterization of different self-assembly process by EIS EIS could provide the interface properties of modified electrodes for each step of immobilization. In EIS, the semi-circle diameter of impedance equals the electron transfer resistance (Ret), which illustrates the electron transfer kinetics of the redox probe at the electrode surface [25]. As shown in Fig. 1, the bare Au electrode displayed a small semi-circle with a Ret of about 220 V (curve a). After electrochemical deposition of AuNPs (curve b), only a straight line was observed in the whole frequency region, which ascribed to the AuNPs and could accelerate the electron transfer. However, the Ret increased when the probe DNA S1 was immobilized on the surface and the value was 60 V (curve c). The semicircle of MCH/ ssDNA/AuNPs/Au increased and the cause was mainly due to negative charge on the surface of the electrode and prevented access of negatively charged ions (curve d). The Ret further increased to 100 V (curve e) when the DNA S1 was hybridized with target DNA S2. The subsequent HCR lead to further increase of Ret (curve f). The
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Fig. 1. EIS after different steps of modification in 5 mM Fe(CN)63/4 and 0.1 M KCl solution with the frequency ranging from 101 to 105 Hz. (a) Au electrode, (b) AuNPs/Au, (c) ssDNA/AuNPs/Au, (d) MCH/ ssDNA/AuNPs/Au, (e) dsDNA/AuNPs/Au, (f) (H2/H1)n/dsDNA/AuNPs/Au, (g) S-ALP/(H2/H1)n/dsDNA/AuNPs/Au.
reason of the above phenomenon was the electrostatic repulsion between the negative-charged phosphate skeletons of DNA and [Fe (CN)6]3/4. Subsequently, the Ret increased to about 880 V after bounded S-ALP, which indicates the formation of S-ALP that was successfully immobilized on the electrode surface. 3.2. Feasibility assay on DNA methylation detection In order to prove the detection feasibility of our developed method on DNA methylation detection and DNA MTase activity assay, the DPV response was recorded when the electrodes were treated with different process. As shown in Fig. 2, a well-defined anodix peak was observed at 0.226 V (curve a), ascribed to the anodix peak of 1-naphthol produced from the hydrolysis of 1naphthyl phosphate catalyzed by ALP. It agreed well with the previous reports on the anodix behavior of 1-naphthol [14]. When the dsDNA was first methylated by M.SssI and then digested by HpaII, the oxidation signal of 1-naphthol was identical with curve a in Fig. 2 (curve b). However, no anodix anodic peak was observed after the un-methylated dsDNA was digested with HpaII endonuclease and then treated with H1, H2 and S-ALP, no anodix anodic
peak was observed in the detection buffer (curve d). It was due to the fact that the dsDNA was specifically recognized and digested by the HpaII endonuclease at the site of 50 -C/CGG-30 , which result in the HCR reaction was prevented and S-ALP could not be bounded the electrode successfully. Moreover, the result indicated that the digestion of S1/S2 hybrids by HpaII endonuclease was blocked by CpG methylation. One-base mismatched DNA S3 was selected to evaluate the cleavage specificity of HpaII endonuclease. The probe S1 was hybridized with S3, and then treated with M.SssI MTase, HpaII, H1, H2 and S-ALP. It was obvious that the oxidation signal of 1naphthol (curve c) was lower than the signal after probe DNA S1 hybridized completely with complementary DNA S2 (curve b), indicating that the hybrid of S1 and S3 can not be recognized by M. SssI MTase because the hybrid S1/S3 does not contain a specific recognition sequence (50 -C/CGG-30 ). These results demonstrated that the developed method could distinguish even one-base mismatched DNA. Therefore, it would be used for highly selective determination of DNA methylation. 3.3. M.SssI MTase activity detection To assess the analytical performance of the M.SssI MTase activity, the dsDNA was methylated with a various concentration of M.SssI (0.02 to 80 unit mL1), digested by HpaII, and then treated with H1, H2 and S-ALP, successively. The DPV response of 1naphthol was recorded in 0.1 M Tris–HCl containing 1 mM Mg (NO3)2 and 1.0 mM 1-naphthyl phosphate. It can be seen that the DPV peaks increased with the increase of M.SssI MTase concentrations (Fig. 3A). The oxidation peak current tended to level off at higher concentration, which might be caused by the almost complete methylation of dsDNA at the high concentration of M.SssI MTase. The DPV current was proportional to the logarithmic value of M.SssI MTase concentration ranging from 0.02 to 50 unit mL1. The linearization equation was Ipa(mA) = 0.2504 log c (unit mL1) + 0.8215 (R = 0.9983) with the detection limit of 0.0067 unit mL1 (inset of Fig. 3B). It is significantly lower than most of the previous reported assays, such as 0.017 unit mL1 [12], 0.02 unit mL1 [26], 0.03 unit mL1 [13], 0.04 unit mL1 [14], 0.05 unit mL1 [18], 0.07 unit mL1 [27], 0.12 unit mL1 [17], 0.3 unit mL1 [28]. 3.4. Assay of the effect of inhibitors on M.SssI MTase activity
Fig. 2. Curve a was the DPV response of dsDNA that was treated with H1, H2 and SALP in 10 mL of detection buffer solution containing 1.0 mM 1-naphthyl phosphate. Curve b was the DPV response of dsDNA first treated with M.SssI MTase and then digested with HpaII and then treated with the same process as curve a. Curve d was the DPV response that the un-methylated dsDNA/AuNPs/Au was treated with HpaII, H1, H2 and S-ALP successively. Curve c was the DPV response that probe DNA S1 was hybridized with single-base mismatched DNA S3, then treated with M.SssI, HpaII, H1, H2 and S-ALP successively.
Recent work has revealed how DNA methylation is linked at the molecular level and DNA MTase anomalies play a direct causal role in tumorigenesis and genetic disease [2,29,30]. Therefore, the investigation of the inhibitory effects of drugs on DNA MTase would be strongly associated with neoplastic development and constitute a key step in carcinogenesis. In this paper, 5-fluorouracil and daunorubicin hydrochloride (daunorubicinHCl) were selected as model inhibitors to investigate DNA MTase activity and methylation level. 5-Fluorouracil is a pyrimidine analog which is used in the treatment of cancer. It is a suicide inhibitor and works through irreversible inhibition of thymidylate synthase. Therefore, the inhibition effect of 5-fluorouracil was investigated in this paper (Fig. 4A). The inhibition ratio increased up to 1.92% with the IC0 value of 139.08 M. DaunorubicinHCl is chemotherapeutic of the anthracycline family that is given as a treatment for some types of cancer. It is most commonly used to treat specific types of leukaemia. As is clear from Fig. 4B, the inhibition efficiency of daunorubicinHCl increased with a rise of daunorubicinHCl concentration and the maximum inhibition efficiency was 49.02%, which was inferior to 5-fluorouracil. These results demonstrated 5-fluorouracil and daunorubicinHCl could inhibit the M.SssI MTase activity variously, which illustrated that the
Z. Xu et al. / Analytica Chimica Acta 829 (2014) 9–14
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Fig. 3. (A) DPV responses of the modified electrodes for different concentrations of M.SssI MTase. The concentrations from a to p were: 0.02, 0.04, 0.06, 0.08, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 unit mL1. (B) The responses current of 1-naphthol with different concentrations of M.SssI MTase from 0.02 to 80 unit mL1. Insert: liner response from 0.02 to 50 unit mL1.
Fig. 4. The inhibition effects of 5-fluorouracil (A), daunorubicinHCl (B) on M.SssI MTase activity, respectively.
developed method could provide a platform for anticancer drugs on DNA MTase. Comparison of these figures are shown in Fig. 5, it turns out that four inhibitors have different effects on M.SssI MTase activity. It also has been found the M.SssI MTase have high selectivity for different inhibitors. 3.5. Effects environmental phenolic endocrine on M.SssI MTase activity Endocrine disruptors are substances that mimic natural hormones in the endocrine system thus cause adverse effects on human and wildlife [31]. Alkylphenols such as bisphenol A (BPA) and nonylphenol are examples of endocrine disruptors. Various
diseases including carcinogenesis may result from the exposure to BPA and nonylphenol. If the M.SssI MTase activity could be influenced by the above typical alkylphenols, it might be a possible carcinogenesis mechanism for alkylphenol endocrine disruptors. For conforming the effects of BPA and nonylphenol on MTase activity, different concentrations of phenols were introduced into 10 mM Tris–HCl (pH 7.40) containing 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, 50% glycerol, 160 mM SAM and 50 unit mL1 M. SssI MTase. Then the dsDNA was treated with HpaII endonucleases and followed by HCR and S-ALP tagging. As shown in Fig. 5A, the anodic currents increased rapidly with increasing the concentration of BPA, which indicated that BPA could improve the M.SssI MTase activity. It can be seen that the promotion ratio of BPA
Fig. 5. The effects of environmental phenolic endocrine: bisphenol A (A), Nonylphenol (B) on M.SssI MTase activity, respectively.
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reached a climax at 200 mM. The maximum promotion efficiency was about 64.41%. Nonylphenol could also improve the M.SssI MTase activity (Fig 5B). The promotion efficiency increased with an increase of nonylphenol concentration and then tended to be stable at 300 mM. The maximum upgrade rate was about 60.66%. These results demonstrated that BPA and nonylphenol could strongly improve the M.SssI MTase activity, which might indicate a kind of possible carcinogenesis mechanism for BPA and nonyl phenol. 4. Conclusion In summary, we have developed an ultrasensitive amplified electrochemical method by coupling the DNA-based HCR. The signal could be amplified by the HCR-based reaction with biotin in the hairpin structures, which could conjugate S-ALP through the specific interaction. Compared with the traditional methods, this method did not need expensive instrument and bisulfite processes with a detection limit of 0.0067 unit mL1. Significantly, the assay approach investigated the different influences on M.SssI MTase of bisphenol A, nonylphenol, 5-fluorouracil and daunorubicin hydrochloride and it could be applied to develop new anti-cancer drugs for the activity of DNA MTase. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21375`079, 21105056) and the Natural Science Foundation of Shandong province, China (Nos. ZR2010BM005, ZR2011BQ001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.04.024. References [1] L. Zhou, X. Cheng, B. Connolly, M. Dickman, P. Hurd, D. Hornby, Journal of Molecular Biology 321 (2002) 591–599.
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DNA-based hybridization chain reaction amplification for assaying the effect of environmental phenolic hormone on DNA methyltransferase activity Zhenning Xu, Huanshun Yin, Yunxiang Han, Yunlei Zhou *, Shiyun Ai * College of Chemistry and Material Science, Shandong Agricultural University, 61 Daizong street, Taian, Shandong 271018, PR China
H I G H L I G H T S
A new electrochemical protocol was fabricated. The DNA-based hybridization chain reaction was used for signal amplification. Fluorouracil and daunorubicin hydrochloride could inhibit the DNA MTase activity. Bisphenol A and nonyl phenol could improve the DNA MTase activity.
G R A P H I C A L A B S T R A C T
?
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2013 Received in revised form 9 April 2014 Accepted 11 April 2014 Available online 24 April 2014
In this work, a novel electrochemical protocol with signal amplification for determination of DNA methylation and methyltransferase activity using DNA-based hybridization chain reaction (HCR) was proposed. After the gold electrode was modified with dsDNA, it was treated with M.SssI MTase, HpaII endonuclease, respectively. And then the HCR was initiated by the target DNA and two hairpin helper DNAs, which lead to the formation of extended dsDNA polymers on the electrode surface. The signal was amplified by the labeled biotin on the hairpin probes. As a result, the streptavidin-alkaline phosphatase (S-ALP) conjugated on the electrode surface through the specific interaction between biotin and S-ALP. ALP could convert 1-naphthyl phosphate into 1-naphthol and the latter could be electrochemically oxidized, which was used to monitor the methylation event and MTase activity. The HCR assay presents good electrochemical responses for the determination of M.SssI MTase at a concentration as low as 0.0067 unit mL1. Moreover, the effects of anti-cancer drug and environmental phenolic hormone on M. SssI MTase activity were also investigated. The results indicated that 5-fluorouracil and daunorubicin hydrochloride could inhibit the activity, and the opposite results were obtained with bisphenol A and nonylphenol. Therefore, this method can not only provide a platform to screen the inhibitors of DNA MTase and develop new anticancer drugs, but also offer a novel technique to investigate the possible carcinogenesis mechanism. ã 2014 Elsevier B.V. All rights reserved.
Keywords: DNA methylation Hybridization chain reaction Methyltransferase Inhibitors Environmental phenolic hormones
1. Introduction
* Corresponding authors. Tel.: +86 538 8249248/7660; fax: +86 538 8242251. E-mail addresses: [email protected] (Y. Zhou), [email protected], [email protected] (S. Ai). http://dx.doi.org/10.1016/j.aca.2014.04.024 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
DNA methylation is the principal driving forces behind the phenomenon of epigenetic [1]. DNA methylation could modulate gene expression and genomic integrity [2], which was catalyzed by methyltransferases (MTase) in the presence of S-adenosyl-L-
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methionine (SAM) as methyl donor. It has been proposed that the ancestral function of DNA methylation could actually restrain the spread of parasitic elements. Aberrant DNA methylation was the most common molecular lesion of the cancer cell [3]. Moreover, aberrant DNA methylation has also been found to be related to the aberrant DNA MTase activity [4,5]. In this regard, we expect to develop a novel sensitive and selective method to detect DNA methylation and DNA MTase activity. Traditional analytical methods such as bisulfites methods [6,7], HPLC (high performance liquid chromatography) [8], fluorescence methods [9,10] and PCR (polymerase chain reaction)-based techniques [11] have been widely used for the determination of DNA methylation. These methods, however, required complicated pretreatment steps, extensive labor resources, and are not applicable for simple and sensitive determination. As known, electrochemical method is the most popular method for DNA methylation and DNA MTase activity with sample preconcentration, time-saving, high sensitivity and selectivity [4,12–15]. Meng et al. has distinguished cytosine and 5-methylcytosine (5-mC) at low potentials by sodium dodecyl sulfate functionalized graphene modified pyrolytic graphite electrode [16]. However, there was an interference caused by thymine (T) because of the almost same oxidation potentials of T and 5-mC [15]. Therefore, direct determination of 5-mC needed further improvement. The DNA MTase and/or restriction enzyme was used in many reports about DNA methylation detection [4,13,17,18]. However, the detection sensitivity needs to be further improved. In 2004, Dirks and Pierce introduced the concept of hybridization chain reaction (HCR). The stable DNA monomers assembled only upon exposure to a target DNA fragment [19]. Tang’s group reported a novel electrochemical immunoassay for the determination of proteins at an ultralow concentration using DNA-based HCR [20]. Chen et al. proposed a strategy based on HCR signal amplification to detect the sequence specific DNA [21]. Compared with traditional construction, HCR could effectively amplify the signal and gain higher sensitivity [22– 24]. Herein, we described an electrochemical method for the detection of DNA methylation and the assay of the
methyltransferase activity based on HCR signal amplification (Scheme 1). After the dsDNA digested by HpaII, the DNA hybrid was cleaved at a specific site 50 -CCGG-30 and the nucleotide sticky end in the target DNA disappeared. However, the cleavage of the HpaII endonuclease was blocked by CpG methylation. When the symmetrical cytosine residues in dsDNA was methylated by M. SssI MTase and digested with HpaII restriction endonuclease, the HCR of DNA initiator strands reacted between biotinylated H1 and H2 hairpin DNA molecules. The streptavidin-alkaline phosphatase (S-ALP) could be conjugated on the electrode surface through the specific interaction between biotin and S-ALP. The ALP molecules could catalytically hydrolyze 1-naphthyl phosphate to produce 1naphthol for generating amplified electrochemical signal. The electrochemical signal was used to quantify the M.SssI MTase activity and screen the inhibitors of M.SssI MTase. 2. Materials and methods 2.1. Reagents Tris(hydroxymethyl) aminomethane (Tris), Tri(2-carboxyethyl) phosphine hydrochloride (TCEP), hydrogen tetrachloroaurate trihydrate (HAuCl43H2O), mercaptopropronic acid (MPA) was obtained from Alfa Aesar (Heysham, Lancashire, UK). Disodium ethylenediaminetetraacetic acid (EDTA), 5-fluorouracil, bisphenol A and nonylphenol were purchased from Aladdin (Shanghai, China). Daunorubicin HCl was purchased from Di Bo Chemical Technology Co., Ltd. (Shanghai, China). S-ALP and PAGE-purified DNA were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The base sequences are as follows: probe DNA (DNA S1): 50 SH-TAG TGT GAT GTC ACC TAG TTG ACC TTC CGG AT-30 , target DNA (DNA S2): 50 -GAA GGA GGG GCG ACT ATC CGG AAG GTC AAC TAG GTG ACA TCA CAC TA-30 , one-base mismatched DNA (DNA S3): 50 GAA GGA GGG GCG ACT ATC CTG AAG GTC AAC TAG GTG ACA TCA CAC TA-30 , hairpin probe H1: (DNA S4): 50 -GGG GCG ACT TGA AAC AGT CGC CCC TCC TTC-biotin-30 , hairpin probe H2: (DNA S5): 50 biotin-GTT TCA AGT CGC CCC GAA GGA GGG GCG ACT-30 . The recognition sites of 50 -CCGG-30 for M.SssI MTase and HpaII
Scheme 1. Schematic illustration of electrochemical method for detection of DNA methylation and assay of DNA MTase activity.
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endonuclease were marked with bold font. The mismatched base T was marked with bold/italic. The synthesized oligonucleotides were diluted in TE buffer to desired stock concentrations and stored at 20 C. M.SssI MTase and restriction endonuclease HpaII are supplied by New England BioLabs (Ipswich, MA) and Fermentas (Maryland, USA), respectively. They were stored at 20 C in a 50 mM Tris–HCl buffer (pH 7.5) containing 10 mM EDTA, 50 mM KCl, 200 mg mL1 bovine serum albumin (BSA), 25% glycerol, 1 mM dithiothreitol (DTT). The buffer solutions for electrochemistry were as follows: Oligonucleotide dissolve buffer (TE buffer): 10 mM Tris–HCl and 1 mM EDTA (pH 8.00); probe immobilization buffer: 10 mM Tris– HCl, 1.0 mM EDTA, 1.0 M NaCl and 1.0 mM TCEP (pH 7.00); DNA hybridization buffer: 10 mM Tris–HCl, 1.0 mM EDTA, and 1.0 M NaCl (pH 7.00); electrochemistry determination buffer: 0.1 M Tris–HCl and 1 mM Mg(NO3)2 (pH 9.80). The pH of buffer solutions was adjusted by adding 0.1 M HCl or 0.1 M NaOH. All reagents were analytically pure grade. All of the solutions and redistilled deionized water were autoclaved. 2.2. Gold nanoparticle deposition A gold electrode was polished carefully to a mirror-like surface with 0.3 mm and 0.05 mm alumina slurry and then sequentially sonicated for 3 min in absolute ethanol and double distilled deionized water, respectively. Prior to the experiment, the bare gold electrode was cyclic-potential scanned within the potential range of 0.2–1.8 V in 0.2 M H2SO4 until a voltammogram characteristic of the clean polycrystalline gold was established. Gold nanoparticle was deposited onto bare gold electrode in an aqueous electrolyte of 3 mM HAuCl4 solution containing 0.1 M KNO3 at a constant potential of 0.2 V for 200 s under stirring. The gold electrodes modified with gold nanoparticles were rinsed with water and dried with N2 for further experiments. The obtained electrode was noted as AuNPs/Au. 2.3. Probe immobilization and hybridization For assembly of probe on AuNPs/Au surface, 5 mL of 0.5 mM thiol-capped probe DNA S1 in probe immobilization buffer was dropped on AuNPs/Au surface and maintained for 12 h at humid conditions. The electrode was thoroughly washed with 10 mM Tris–HCl (pH 7.40) for three times. Then 5 mL Tris–HCl (10 mM, pH 7.40) containing 3 mM MCH was dropped on the electrode surface and kept for 1 h to eliminate the un-immobilized probe DNA S1 for its good recognition ability. Thus, the ssDNA/AuNPs/Au was obtained. The hybridization experiment was performed by dripping 5 mL DNA hybridization buffer containing 0.5 mM of target DNA S2 on the electrode surface and the hybridization process was kept at 37 C for 2 h. After that, the electrode was rinsed three times with 10 mM Tris–HCl (pH 7.40) to remove the un-hybridized target DNA and dried with nitrogen blowing. The obtained electrode was named as dsDNA/AuNPs/Au. 2.4. Methylation and cleavage For methylation process, the dsDNA was treated with 10 mM Tris–HCl buffer (pH 7.50) containing 160 mM SAM, 50 mM KCl, 10 mM EDTA, 200 mg mL1 BSA, 25% glycerol, 1 mM DTT and various concentrations of M.SssI (from 0 to 80 unit mL1 at 37 C for 2 h. Then, the obtained electrode was rinsed with 10 mM Tris–HCl (pH 7.40) for three times. And then, the above electrode was digested by HpaII endonucleases at 37 C in 10 mM Tris–HCl buffer (pH 7.50) containing 50 unit mL1 HpaII, 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, and 50% glycerol for 2 h. After
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digestion, the electrode was thoroughly washed with 10 mM Tris– HCl (pH 7.40). 2.5. HCR and S-ALP tagging After washing with Tris–HCl, the modified electrode was incubated with 5 mL hybridization buffer containing biotinylated hairpin probe H1 (2.5 mM) and H2 (2.5 mM) for 3 h. The electrode was thoroughly washed with 10 mM Tris–HCl (pH 7.40) and the obtained electrode was named as (H2/H1)n/dsDNA/AuNPs/Au. After that, 5 mL S-ALP was soaked on the electrode surface for 30 min at room temperature. The electrode was then rinsed three times with 10 mM Tris–HCl buffer (pH 7.40) and dried at room temperature and was noted as S-ALP/(H2/H1)n/dsDNA/AuNPs/Au. 2.6. Inhibition the activity of M.SssI MTase To further study the activity of M.SssI, 5-fluorouracil, daunorubicin HCl, nonylphenol and bisphenol A were used to the model inhibitors. The DNA S1/S2 hybrids were performed at 37 C for 2 h in 10 mM Tris–HCl (pH 7.40) containing 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, 50% glycerol, 160 mM SAM, 50 unit mL1 M.SssI and various concentration of inhibitors. The inhibition efficiency (%) was estimated as follows: Inhibitionð%Þ ¼
I I1 I
where I is the peak current of 1-naphthol obtained after the S1/S2 hybrids successively treated with M.SssI, HpaII, H1, H2 and S-ALP. I1 is the inhibited peak current of 1-naphthol. 2.7. Electrochemical determination The electrode was dipped into a stirring Tris–HCl containing 1 mM Mg(NO3)2 (pH 9.80) and 1.0 mM 1-naphthyl phosphate for 3 min. Differential pulse voltammetry (DPV) was performed with a CHI 660C electrochemical workstation (Austin, USA). The measurements were based on a conventional three-electrode system. The parameters are 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, 6 s. Electrochemical impedance spectroscopy (EIS) was performed with a CHI 660C electrochemical workstation in 5 mM [Fe(CN)6]3/4 (1:1) solution containing 0.1 M KCl as the supporting electrolyte. The frequency was ranged from 101 to 105 Hz. 3. Results and discussion 3.1. Characterization of different self-assembly process by EIS EIS could provide the interface properties of modified electrodes for each step of immobilization. In EIS, the semi-circle diameter of impedance equals the electron transfer resistance (Ret), which illustrates the electron transfer kinetics of the redox probe at the electrode surface [25]. As shown in Fig. 1, the bare Au electrode displayed a small semi-circle with a Ret of about 220 V (curve a). After electrochemical deposition of AuNPs (curve b), only a straight line was observed in the whole frequency region, which ascribed to the AuNPs and could accelerate the electron transfer. However, the Ret increased when the probe DNA S1 was immobilized on the surface and the value was 60 V (curve c). The semicircle of MCH/ ssDNA/AuNPs/Au increased and the cause was mainly due to negative charge on the surface of the electrode and prevented access of negatively charged ions (curve d). The Ret further increased to 100 V (curve e) when the DNA S1 was hybridized with target DNA S2. The subsequent HCR lead to further increase of Ret (curve f). The
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Fig. 1. EIS after different steps of modification in 5 mM Fe(CN)63/4 and 0.1 M KCl solution with the frequency ranging from 101 to 105 Hz. (a) Au electrode, (b) AuNPs/Au, (c) ssDNA/AuNPs/Au, (d) MCH/ ssDNA/AuNPs/Au, (e) dsDNA/AuNPs/Au, (f) (H2/H1)n/dsDNA/AuNPs/Au, (g) S-ALP/(H2/H1)n/dsDNA/AuNPs/Au.
reason of the above phenomenon was the electrostatic repulsion between the negative-charged phosphate skeletons of DNA and [Fe (CN)6]3/4. Subsequently, the Ret increased to about 880 V after bounded S-ALP, which indicates the formation of S-ALP that was successfully immobilized on the electrode surface. 3.2. Feasibility assay on DNA methylation detection In order to prove the detection feasibility of our developed method on DNA methylation detection and DNA MTase activity assay, the DPV response was recorded when the electrodes were treated with different process. As shown in Fig. 2, a well-defined anodix peak was observed at 0.226 V (curve a), ascribed to the anodix peak of 1-naphthol produced from the hydrolysis of 1naphthyl phosphate catalyzed by ALP. It agreed well with the previous reports on the anodix behavior of 1-naphthol [14]. When the dsDNA was first methylated by M.SssI and then digested by HpaII, the oxidation signal of 1-naphthol was identical with curve a in Fig. 2 (curve b). However, no anodix anodic peak was observed after the un-methylated dsDNA was digested with HpaII endonuclease and then treated with H1, H2 and S-ALP, no anodix anodic
peak was observed in the detection buffer (curve d). It was due to the fact that the dsDNA was specifically recognized and digested by the HpaII endonuclease at the site of 50 -C/CGG-30 , which result in the HCR reaction was prevented and S-ALP could not be bounded the electrode successfully. Moreover, the result indicated that the digestion of S1/S2 hybrids by HpaII endonuclease was blocked by CpG methylation. One-base mismatched DNA S3 was selected to evaluate the cleavage specificity of HpaII endonuclease. The probe S1 was hybridized with S3, and then treated with M.SssI MTase, HpaII, H1, H2 and S-ALP. It was obvious that the oxidation signal of 1naphthol (curve c) was lower than the signal after probe DNA S1 hybridized completely with complementary DNA S2 (curve b), indicating that the hybrid of S1 and S3 can not be recognized by M. SssI MTase because the hybrid S1/S3 does not contain a specific recognition sequence (50 -C/CGG-30 ). These results demonstrated that the developed method could distinguish even one-base mismatched DNA. Therefore, it would be used for highly selective determination of DNA methylation. 3.3. M.SssI MTase activity detection To assess the analytical performance of the M.SssI MTase activity, the dsDNA was methylated with a various concentration of M.SssI (0.02 to 80 unit mL1), digested by HpaII, and then treated with H1, H2 and S-ALP, successively. The DPV response of 1naphthol was recorded in 0.1 M Tris–HCl containing 1 mM Mg (NO3)2 and 1.0 mM 1-naphthyl phosphate. It can be seen that the DPV peaks increased with the increase of M.SssI MTase concentrations (Fig. 3A). The oxidation peak current tended to level off at higher concentration, which might be caused by the almost complete methylation of dsDNA at the high concentration of M.SssI MTase. The DPV current was proportional to the logarithmic value of M.SssI MTase concentration ranging from 0.02 to 50 unit mL1. The linearization equation was Ipa(mA) = 0.2504 log c (unit mL1) + 0.8215 (R = 0.9983) with the detection limit of 0.0067 unit mL1 (inset of Fig. 3B). It is significantly lower than most of the previous reported assays, such as 0.017 unit mL1 [12], 0.02 unit mL1 [26], 0.03 unit mL1 [13], 0.04 unit mL1 [14], 0.05 unit mL1 [18], 0.07 unit mL1 [27], 0.12 unit mL1 [17], 0.3 unit mL1 [28]. 3.4. Assay of the effect of inhibitors on M.SssI MTase activity
Fig. 2. Curve a was the DPV response of dsDNA that was treated with H1, H2 and SALP in 10 mL of detection buffer solution containing 1.0 mM 1-naphthyl phosphate. Curve b was the DPV response of dsDNA first treated with M.SssI MTase and then digested with HpaII and then treated with the same process as curve a. Curve d was the DPV response that the un-methylated dsDNA/AuNPs/Au was treated with HpaII, H1, H2 and S-ALP successively. Curve c was the DPV response that probe DNA S1 was hybridized with single-base mismatched DNA S3, then treated with M.SssI, HpaII, H1, H2 and S-ALP successively.
Recent work has revealed how DNA methylation is linked at the molecular level and DNA MTase anomalies play a direct causal role in tumorigenesis and genetic disease [2,29,30]. Therefore, the investigation of the inhibitory effects of drugs on DNA MTase would be strongly associated with neoplastic development and constitute a key step in carcinogenesis. In this paper, 5-fluorouracil and daunorubicin hydrochloride (daunorubicinHCl) were selected as model inhibitors to investigate DNA MTase activity and methylation level. 5-Fluorouracil is a pyrimidine analog which is used in the treatment of cancer. It is a suicide inhibitor and works through irreversible inhibition of thymidylate synthase. Therefore, the inhibition effect of 5-fluorouracil was investigated in this paper (Fig. 4A). The inhibition ratio increased up to 1.92% with the IC0 value of 139.08 M. DaunorubicinHCl is chemotherapeutic of the anthracycline family that is given as a treatment for some types of cancer. It is most commonly used to treat specific types of leukaemia. As is clear from Fig. 4B, the inhibition efficiency of daunorubicinHCl increased with a rise of daunorubicinHCl concentration and the maximum inhibition efficiency was 49.02%, which was inferior to 5-fluorouracil. These results demonstrated 5-fluorouracil and daunorubicinHCl could inhibit the M.SssI MTase activity variously, which illustrated that the
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Fig. 3. (A) DPV responses of the modified electrodes for different concentrations of M.SssI MTase. The concentrations from a to p were: 0.02, 0.04, 0.06, 0.08, 0.1, 0.5, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80 unit mL1. (B) The responses current of 1-naphthol with different concentrations of M.SssI MTase from 0.02 to 80 unit mL1. Insert: liner response from 0.02 to 50 unit mL1.
Fig. 4. The inhibition effects of 5-fluorouracil (A), daunorubicinHCl (B) on M.SssI MTase activity, respectively.
developed method could provide a platform for anticancer drugs on DNA MTase. Comparison of these figures are shown in Fig. 5, it turns out that four inhibitors have different effects on M.SssI MTase activity. It also has been found the M.SssI MTase have high selectivity for different inhibitors. 3.5. Effects environmental phenolic endocrine on M.SssI MTase activity Endocrine disruptors are substances that mimic natural hormones in the endocrine system thus cause adverse effects on human and wildlife [31]. Alkylphenols such as bisphenol A (BPA) and nonylphenol are examples of endocrine disruptors. Various
diseases including carcinogenesis may result from the exposure to BPA and nonylphenol. If the M.SssI MTase activity could be influenced by the above typical alkylphenols, it might be a possible carcinogenesis mechanism for alkylphenol endocrine disruptors. For conforming the effects of BPA and nonylphenol on MTase activity, different concentrations of phenols were introduced into 10 mM Tris–HCl (pH 7.40) containing 0.1 mM EDTA, 1 mM DTT, 200 mg mL1 BSA, 50% glycerol, 160 mM SAM and 50 unit mL1 M. SssI MTase. Then the dsDNA was treated with HpaII endonucleases and followed by HCR and S-ALP tagging. As shown in Fig. 5A, the anodic currents increased rapidly with increasing the concentration of BPA, which indicated that BPA could improve the M.SssI MTase activity. It can be seen that the promotion ratio of BPA
Fig. 5. The effects of environmental phenolic endocrine: bisphenol A (A), Nonylphenol (B) on M.SssI MTase activity, respectively.
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reached a climax at 200 mM. The maximum promotion efficiency was about 64.41%. Nonylphenol could also improve the M.SssI MTase activity (Fig 5B). The promotion efficiency increased with an increase of nonylphenol concentration and then tended to be stable at 300 mM. The maximum upgrade rate was about 60.66%. These results demonstrated that BPA and nonylphenol could strongly improve the M.SssI MTase activity, which might indicate a kind of possible carcinogenesis mechanism for BPA and nonyl phenol. 4. Conclusion In summary, we have developed an ultrasensitive amplified electrochemical method by coupling the DNA-based HCR. The signal could be amplified by the HCR-based reaction with biotin in the hairpin structures, which could conjugate S-ALP through the specific interaction. Compared with the traditional methods, this method did not need expensive instrument and bisulfite processes with a detection limit of 0.0067 unit mL1. Significantly, the assay approach investigated the different influences on M.SssI MTase of bisphenol A, nonylphenol, 5-fluorouracil and daunorubicin hydrochloride and it could be applied to develop new anti-cancer drugs for the activity of DNA MTase. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21375`079, 21105056) and the Natural Science Foundation of Shandong province, China (Nos. ZR2010BM005, ZR2011BQ001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.04.024. References [1] L. Zhou, X. Cheng, B. Connolly, M. Dickman, P. Hurd, D. Hornby, Journal of Molecular Biology 321 (2002) 591–599.
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