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A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4
Analytica Chimica Acta xxx (2016) 1e6
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A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4 Pei Liu, Kexin Zhang, Ranran Zhang, Huanshun Yin, Yunlei Zhou**, Shiyun Ai* College of Chemistry and Material Science, Shandong Agricultural University, Taian, 271018, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel colorimetrical strategy was fabricated. DNA MTase activity was detected using this biosensor. The color change was monitored by the keypad lock of duplex DNA modified meso-SiO2@Fe3O4. Real biological matrices and inhibitors screening were performed by the biosensor.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2016 Received in revised form 8 March 2016 Accepted 14 March 2016 Available online xxx
Abnormal level of DNA methyltransferase (MTase) e mediated DNA methylation is closely related with cancer and bacterial diseases. Herein, a novel strategy based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4 was developed for colorimetric assay of Dam MTase activity. When the Dam MTase was introduced, the duplex DNA can be methylated at a palindrome sequence of 50 -GATC-30 and cleaved by the methylation-sensitive restriction endonuclease Dpn I. Due to the instability of the newly formed DNA fragment, the hybrid will separated into a single-stranded DNA. Then the keypad lock will open, and the catalytic reaction of TMB and H2O2 can be initiated through the pores of meso-SiO2@Fe3O4, and a high color signal can be clearly observed by the naked eye. Contrarily, without Dam MTase, the catalytic reaction will not be initiated, and result no color signal. The proposed method exhibited a wide dynamic range with a low detection limit of 0.73 U/mL. Additionally, this way can be performed in human serum with satisfying recovery. And the inhibition of Dam MTase can also be well demonstrated by using paclitaxel as a model. Therefore, the designed way not only provides a platform for monitoring Dam MTase activity, but also useful for further application in disease diagnosis and drug discovery. © 2016 Published by Elsevier B.V.
Keywords: DNA methyltransferase meso-SiO2@Fe3O4 Paclitaxel Colorimetric assay
1. Introduction DNA methylation, an important epigenetic event [1], has
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Zhou), [email protected] (S. Ai).
received a large amount of attention due to its close relationship with lots of biological processes, such as DNA repair, gene transcription, and embryogenesis [2]. It refers to the process that a methyl group from S-adenosyl-L-methionine (SAM) transferred to adenine or cytosine in the target DNA under the catalytic of DNA methyltransferase (MTase) [3]. In recent years, more and more studies reported that abnormal level of DNA MTase would set off aberrant DNA methylation [4], which has profound implications in
http://dx.doi.org/10.1016/j.aca.2016.03.028 0003-2670/© 2016 Published by Elsevier B.V.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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some critical diseases such as cancer and bacterial diseases [5]. Thus, the detection of DNA MTase activity has aroused an increasing interest over the past years. In recent years, traditional assay of DNA MTase activity including radioactive labeling [6], polymerase chain reaction (PCR) [7,8], and high-performance liquid chromatography (HPLC) [9] have been established. However, these methods are time-consuming, laborious, expensive and not sensitive. In order to improve the shortcomings, electrochemical biosensors [10,11], chemiluminescence ways [12], fluorescence methods [13], photoelectrochemistry [14] and colorimetric assay [15] are developed to detect the activity of DNA MTase. Owing to the advantages of rapidity, simplicity, cost-effectiveness, and especially easy to read out by the naked eye, colorimetric assay has received an intensive attention. For example, Jiang's group [16] fabricated a colorimetric biosensor for DNA MTase detection based on MTase-protection of the DNA-gold nanoparticles (AuNPs). Li et al. [17] developed a novel strategy for detection of DNA MTase activity based on methylationresponsive DNAzyme with the detection limit of 6 U/mL. To amplify the signal, a label-free colorimetric method for DNA MTase assay based on methylation-blocked cascade amplification was established by Zhao et al. [18]. However, the instability of the AuNPs and the complex reaction conditions of enzymes may limit the use of the above methods. Therefore, a method that is more stable and simpler is still needed. Laterly, we have reported a novel electrochemical biosensors based on DNA functionalized nano mesoporous silica (MSNs) for assay of the DNA MTase activity [19]. Inspired by the unique feature of MSNs, such as biocompatibility, tunable pore structure, high thermal stability and large surface area, and the catalytic activity of Fe3O4, we developed a simple colorimetric approach for detection of DNA MTase activity based on the keypad lock of duplex DNA
modified meso-SiO2@Fe3O4. Owing to the report that the pore of the nano mesoporous silica can be blocked by a DNA duplex [20], the DNA MTase activity can be monitored by the color change of methylation-cleaved initiated catalytic reaction. Additionally, this way can be performed in human serum with satisfying recovery, and used for screening the antimicrobial drugs with high selectivity. 2. Experimental 2.1. Reagents and materials Tetraethylorthosilicate (TEOS), (3-aminopropyl) trimethoxysilane (APTES), 1-[3-(dimethylamino) propyl]-3ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosucnimide sodium salt (NHS), 3,30 ,5,50 -Tetramethylbenzidine (TMB), tris (hydroxymethyl) aminomethane (Tris), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Aladdin (Shanghai, China). N-cetyltrimethylammonium bromide (CTAB) was obtained from Xinran Industrial Co., Ltd (Shanghai, China). Paclitaxel was purchased from Dibo Chemical Technology Co., Ltd. (Shanghai, China). FeSO4$7H2O, Fe2(SO4)3, H2O2, and other chemical reagents were all purchased from Kay Tong Chemical Reagents Co., Ltd (Tianjin, China). The serum samples were provided by the university hospital. All reagents employed were of analytical grade and the solutions were prepared by deioned water after high pressure steam sterilization. The Dam MTase, SAM and Dpn I endonuclease were supplied by New England Biolabs (Ipswich, MA) and Fermentas (MD, USA). According to the supplier, Dam MTase and restriction endonuclease Dpn I were stored at 20 C in the refrigerator in Dam MTase store buffer (50 mM KCl, 50 mM TriseHCl, 10 mM EDTA, 1 mM dithiothreitol, 200 mg/mL BSA and 50% glycerol, pH 7.5) and Dpn I store buffer (10 mM TriseHCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 500 mg/mL BSA, and 50% Glycerol, pH 7.4), respectively. The DNA were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China) and stored in the TE buffer (10 mM TriseHCl, and 1 mM EDTA, pH 8.0) at 20 C in the refrigerator. The base sequences are as follows: DNA S1: 50 - COOH - ATAGT GATC ATTGTTATTAGGGAG - 30 DNA S2: 50 - CTCCCTAATAACAAT GATC ACTAT - 30 UVeVis absorption spectra was carried out on a UV-2450 Shimadzu Vis-spectrometer (Japan).Transmission electron microscopy (TEM) was performed on a JEM-100CX electron microscope (Japan). Fourier transform infrared (FTIR) spectra were obtained on a Thermo Nicolet-380 IR spectrophotometer (USA). 2.2. Synthesis of meso-SiO2@Fe3O4
Scheme 1. Schematic illustration of the Dam MTase activity colorimetric assay.
Briefly [21,22], a clear solution (150 mL) containing 5.6 mM
Fig. 1. TEM image of meso-SiO2@Fe3O4 (A) and (B). Photographs of meso-SiO2@Fe3O4 (C) and meso-SiO2@Fe3O4 attracted towards the magnet for 1 min (D).
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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FeSO4$7H2O and 11.2 mM Fe2(SO4)3 was heated to 50 C with the protection of N2, followed by the addition of 12.5 mL concentrated ammonia aqueous solution (NH3$H2O) under vigorous stirring. After 30 min, the black products were washed three times with water and dried at 60 C for 6 h. Then 0.1 g as-prepared Fe3O4 was mixed with 0.3 g CTAB, 1.0 g NH3$H2O, 80 mL deioned water and 60 mL ethanol, and stirred vigorously for 30 min. Subsequently, TEOS (0.4 g) was added dropwise to the mixture. After stirring for 2 h, the obtained products were collected with a magnet and washed with water and ethanol. In order to remove the template CTAB, the purified products were redispersed in 60 mL of methanol to refluxe for 48 h at 80 C. The extraction was repeated for three times, and the products were washed with ethanol. Finally, the products were redispersed in a mixture of 100 mL ethanol and 7 mL APTES. After ‘24 h’ stirring, the meso-SiO2@Fe3O4 were washed with deioned water and dried. FT-IR was carried out to confirm the existence of amine group on meso-SiO2@Fe3O4 (Fig. S1 in the Supporting Information). 2.3. Assembly of DNA DNA S1 (10 mL) was first activated by 50 mL of TriseHCl (pH 7.4) containing EDC (0.5 mg/mL) and NHS (0.5 mg/mL) for 2 h. Then 10 mL of meso-SiO2@Fe3O4 (4 g/L) was added and reacted for another 2 h. After magnetic separation, the meso-SiO2@Fe3O4-DNA S1 was washed three times with TE buffer and dispersed in 50 mL of solution containing 10 mM TriseHCl (pH 7.0), 1.0 mM EDTA and 1.0 M NaCl, followed the addition of 10 mL of DNA S2. Next, the mixture was incubated at 37 C for 2 h to form meso-SiO2@Fe3O4DNA S1-S2. Finally, the products were separated and washed, then saved in 10 mL of TE buffer. The UVevis spectroscopy was carried out to characterize that DNA have been assembled to mesoSiO2@Fe3O4 (Fig. S2). 2.4. Colorimetric assay of Dam MTase activity In a typical experiment, the methylation and cleavage was carried out by adding various concentration of Dam MTase, 80 mM
Fig. 2. UVeVis absorption spectra of different sample. (a) TMB and H2O2, (b) mesoSiO2@Fe3O4, TMB and H2O2, (c) meso-SiO2@Fe3O4-DNA S1, TMB and H2O2, (d) mesoSiO2@Fe3O4-DNA S1-S2, TMB and H2O2, (e) meso-SiO2@Fe3O4-DNA S1-S2, Dpn I, TMB and H2O2, (f) meso-SiO2@Fe3O4-DNA S1-S2, Dam MTase, Dpn I, TMB and H2O2. The corresponding photograph was also presented, respectively.
Fig. 3. (A) The relative UVeVis absorbance intensities catalyzed by meso-SiO2@Fe3O4 fixed with different concentration of DNA. (B) The relative UVeVis absorbance intensities to various catalytic reaction time. (C) The relative UVeVis absorbance intensities to different time of methylation reaction and cleaving. The Dam MTase concentration in all figures is 40 U/mL. Error bars show the standard deviation of three experiments.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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SAM, 1 Dam buffer (50 mM TriseHCl, 10 mM EDTA, 5 mM 2mercaptoethanol, pH 7.5), 50 U/mL Dpn I and 1 NEB buffer (20 mM Tris-acetate, 50 mM Potassium acetate, 10 mM magnesium acetate and 100 mg/mL BSA, pH 7.9) to 10 mL of TE buffer containing SiO2@Fe3O4-DNA S1-S2. The mixture was incubated at 37 C for 2 h. Then a magnet was used to collect the sediment. After washing with vast PBS buffer (0.1 M, pH 7.0), 90 mL of 0.1 M PBS buffer (pH 5.0), 20 mL of TMB (8 mM) and 40 mL of H2O2 (100 mM) was added successively. The absorption spectra were recorded in 20 min by UVeVis spectrometer. 2.5. Influence of paclitaxel on Dam MTase activity To investigate the effect of paclitaxel on Dam MTase activity, the similar procedures were performed with mentioned above, except adding of different concentrations of the inhibitor together with Dam MTase. 3. Results and discussion
color change from colorless to blue. At higher activity of Dam MTase, more DNA hybrid was methylated and cleaved, and more Fe3O4 were exposed, and thus, a stronger color change was obtained. In this way, by the naked eye or with simple UVeVis spectroscopy, we could detect the activity of Dam MTase quantitatively. 3.2. Characterization of meso-SiO2@Fe3O4 Fig. 1A depicted the TEM image of the meso-SiO2@Fe3O4 nanosphere with average diameter of 400 nm. It could be observed that the Fe3O4 core was well coated by a silica shell with thickness of about 30 nm. Moreover, abundant of mesopores in the silica shell could be obtained from HRTEM image (Fig. 1B). It demonstrated that the meso-SiO2@Fe3O4 have been successfully synthesized, which was consistent with previous work [25]. Additionally, the magnetic separability of meso-SiO2@Fe3O4 was supported by the experimental observation that the nanospheres in solution were attracted towards the magnet within 1 min (Fig. 1D), which could be redispersed again like Fig. 1C.
3.1. Principle of the colorimetric assay 3.3. The feasibility of the designed system The developed colorimetric strategy in profiling the MTase activity is described in Scheme 1. Primarily, the synthesized mesoSiO2@Fe3O4 was first assembled with DNA S1 through the reaction between the amino-group and carboxyl. After hybridization, the DNA duplex can serve as a cap to block pores of the meso-SiO2@Fe3O4 to prevent the contact of guest molecules with Fe3O4. Then a weak color change could be generated. Subsequently, in the presence of Dam MTase and SAM, the DNA hybrid was methylated. Since Dpn I is a methylation-sensitive restriction endonuclease, the methylated DNA hybrid can be cleaved at a specific site of 50 GAmTC e 30 [23] and formed a new hybrid. However, the new hybrid is unstable [24] and easy to separate into a single-stranded DNA fragment. Therefore, the lock is open and the colorimetric reaction of TMB and H2O2 is preformed by Fe3O4, who has been proved to be an excellent mimetic enzyme, resulting a significant
To demonstrate the feasibility of the designed system, several experiments were carried out. As shown in Fig. 2, characteristic absorption peaks at 652 nm for the oxidized TMB product could be observed. Because colorless TMB can be oxidized by H2O2 to yield a blue colored product, which can be recorded by the absorption spectra. To test the peroxidase-like activity of meso-SiO2@Fe3O4, catalytic oxidation of TMB in the presence of H2O2 was performed. Compare to the blank sample (curve a), the peak (curve b) increased significantly due to the catalytic of Fe3O4. When meso-SiO2@Fe3O4 first connected with DNA S1, the peak (curve c) remained. However, the absorbance (curve d) decreased after DNA hybridization, which proved that the DNA duplex can block pores of the meso-SiO2@Fe3O4 to prevent the contact of guest molecules with Fe3O4. Moreover, in the presence of Dam MTase and Dpn I, the significant
Fig. 4. (A) Photograph and (B) absorption spectra in the presence of different concentrations of Dam MTase. (C) Linear relationship between the absorption intensity and the Dam MTase concentration.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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performance was the reaction time. In this method, a high colorimetric signal was expected to be achieved by a long time of catalytic reaction. The investigated results indicated that the absorbance intensity increased with the increase of catalytic reaction time (Fig. 3B). And 20 min was chosen for the following experiments due to the highest signal. Then, the time of methylation reaction and cleaving catalyzed by Dam MTase and Dpn I was optimized and the result was given in Fig. 3C. 120 min was selected because a plateau effect was reached after this time. 3.5. Methylation assay
Fig. 5. Selectivity of the designed strategy. Both of the concentrations of Dam MTase and M.SssI were 40 U/mL.
enhancement (curve f) showed the successful methylation and cleave of the hybridized DNA to form a new single-stranded DNA. On the contrary, without Dam MTase in the system, curve e was the same as curve d, indicating the unmethylated hybrid DNA cannot be cleaved by Dpn I. Besides, these results can also be estimated by the change of the color from colorless to various kinds of blue. Therefore, the sensitive characterization of Dam MTase activity can be successfully performed by this colorimetric biosensor.
To investigate the analytical performance of the designed strategy, Dam MTase at various concentrations were performed under the optimal conditions. As seen in Fig. 4A (left to right), the color changed from colorless to blue with the increasing of the Dam MTase concentrations. The corresponding UVevis absorption spectra were shown in Fig. 4B. As the concentration of Dam MTase increased, the absorption intensity increased. This was consistent with the fact that the more DNA hybrid was methylated and cleaved, the more Fe3O4 were exposed, and thus, a stronger color change can be obtained. Additionally, a good linear curve was gained between the absorption intensity and concentration of Dam MTase from 0 to 40 U/mL (Fig. 4C). With a correlation coefficient of 0.998, the regression equation was A ¼ 0.0092 c (U/mL) þ 0.068. The limit of detection of Dam MTase was approximately 0.73 U/mL in terms of the rule of 3s/k, which was compared with previously reported assays (Table S1 in the Supporting Information). 3.6. Selectivity study
3.4. Optimization of assay system To achieve the best analytical performance, the assay conditions were first optimized. Fig. 3A showed the absorbance of oxidized TMB, which was catalyzed by meso-SiO2@Fe3O4 fixed with various concentration of DNA. The colorimetric signal was declined as the concentration of DNA increased, and it reached a minimum at 10 mM. As a result, 10 mM was selected as the optimum concentration. The other significant factor that influence the sensing
Fig. 6. Absorption intensities obtained from the testing of TriseHCl buffer (blue column) and human serum sample (red column) spiked with Dam MTase. The concentrations of Dam MTase were 0 (control), 1, 10 and 40 U/mL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Another factor to assess the performance of the method is selectivity. In order to evaluated, M. SssI was selected as an interference enzyme. As can be seen from Fig. 5, a significant enhancement of absorption intensity was obtained in the presence of Dam MTase. On the contrary, no distinct was observed in the presence of M. SssI. Because the recognition of the interference enzyme was 50 - CG -30 sequence which was different from that of Dam MTase. These results indicated that this method can easily discriminate Dam MTase from M.SssI MTase. Therefore, the designed strategy for Dam MTase activity detection possessed a good selectivity.
Fig. 7. Inhibition efficiency of Dam MTase by paclitaxel.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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3.7. Dam MTase sensing in human serum A significant challenge for assay of the MTase activity is to achieve the detection in complex biological sample. In this method, we investigated the standardized human serum sample spiked with Dam MTase at three different concentrations of Dam MTase. The human serum samples were diluted in 1:10 ratio by 50 mM TriseHCl buffer. As can be seen in Fig. 6, the absorbance intensity from the diluted human serum sample was well consistent with that of from TriseHCl buffer. The recoveries were in the range of 90.2%e102.5% with a maximum RSD of 5.62%. These results indicated that the designed method held a great promise application in high concentrations of interfering species and further potential for the practicality in complex biological samples. 3.8. Assay of the inhibition of Dam MTase activity To further extend the potential application of the designed biosensor, paclitaxel, an anticancer and antimicrobial drug, was chosen as a model to investigate the influence of inhibitors on DNA methylation. As shown in Fig. 7, increasing the inhibitor concentration, the inhibition efficiency gradually increased. The paclitaxel was found to inhibit 67.1% Dam MTase activity. In addition, the IC50 value (the inhibitor concentration required to reduce enzyme activity by 50%) was considered to be about 170 mM, which is similar to the previous report [26]. These results indicated that paclitaxel could inhibit the Dam MTase activity strongly. So the method could be used to screen inhibitors of Dam MTase. 4. Conclusion In conclusion, we have successfully developed a novel colorimetric assay for Dam MTase activity, using the control of the opening or closing of the meso-SiO2@Fe3O4 pores by DNA, which have an impact on the catalytic of TMB and H2O2. As a result, the asproposed biosensor offered a highly sensitive method for detection of Dam MTase activity with a wide range and a low detection of 0.73 U/mL. Importantly, this way can be performed in human serum with satisfying recovery, and used for screening the antimicrobial drugs with high selectivity. By exchanging the corresponding DNA sequence, the method could be extended to other DNA MTase detection such as cancer-related DNA MTase family and the corresponding inhibitor screening. Therefore, this method was expected to be useful for further application in disease diagnosis and drug development with these attractive analytical characteristics. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21375079, 21105056), the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109) and the Natural Science Foundation of Shandong province China (No. ZR2014BQ029). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2016.03.028. References
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[1] A. Jeltsch, R.Z. Jurkowska, T.P. Jurkowski, K. Liebert, P. Rathert, M. Schlickenrieder, Application of DNA methyltransferases in targeted DNA
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4 Pei Liu, Kexin Zhang, Ranran Zhang, Huanshun Yin, Yunlei Zhou**, Shiyun Ai* College of Chemistry and Material Science, Shandong Agricultural University, Taian, 271018, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A novel colorimetrical strategy was fabricated. DNA MTase activity was detected using this biosensor. The color change was monitored by the keypad lock of duplex DNA modified meso-SiO2@Fe3O4. Real biological matrices and inhibitors screening were performed by the biosensor.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 January 2016 Received in revised form 8 March 2016 Accepted 14 March 2016 Available online xxx
Abnormal level of DNA methyltransferase (MTase) e mediated DNA methylation is closely related with cancer and bacterial diseases. Herein, a novel strategy based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4 was developed for colorimetric assay of Dam MTase activity. When the Dam MTase was introduced, the duplex DNA can be methylated at a palindrome sequence of 50 -GATC-30 and cleaved by the methylation-sensitive restriction endonuclease Dpn I. Due to the instability of the newly formed DNA fragment, the hybrid will separated into a single-stranded DNA. Then the keypad lock will open, and the catalytic reaction of TMB and H2O2 can be initiated through the pores of meso-SiO2@Fe3O4, and a high color signal can be clearly observed by the naked eye. Contrarily, without Dam MTase, the catalytic reaction will not be initiated, and result no color signal. The proposed method exhibited a wide dynamic range with a low detection limit of 0.73 U/mL. Additionally, this way can be performed in human serum with satisfying recovery. And the inhibition of Dam MTase can also be well demonstrated by using paclitaxel as a model. Therefore, the designed way not only provides a platform for monitoring Dam MTase activity, but also useful for further application in disease diagnosis and drug discovery. © 2016 Published by Elsevier B.V.
Keywords: DNA methyltransferase meso-SiO2@Fe3O4 Paclitaxel Colorimetric assay
1. Introduction DNA methylation, an important epigenetic event [1], has
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Zhou), [email protected] (S. Ai).
received a large amount of attention due to its close relationship with lots of biological processes, such as DNA repair, gene transcription, and embryogenesis [2]. It refers to the process that a methyl group from S-adenosyl-L-methionine (SAM) transferred to adenine or cytosine in the target DNA under the catalytic of DNA methyltransferase (MTase) [3]. In recent years, more and more studies reported that abnormal level of DNA MTase would set off aberrant DNA methylation [4], which has profound implications in
http://dx.doi.org/10.1016/j.aca.2016.03.028 0003-2670/© 2016 Published by Elsevier B.V.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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some critical diseases such as cancer and bacterial diseases [5]. Thus, the detection of DNA MTase activity has aroused an increasing interest over the past years. In recent years, traditional assay of DNA MTase activity including radioactive labeling [6], polymerase chain reaction (PCR) [7,8], and high-performance liquid chromatography (HPLC) [9] have been established. However, these methods are time-consuming, laborious, expensive and not sensitive. In order to improve the shortcomings, electrochemical biosensors [10,11], chemiluminescence ways [12], fluorescence methods [13], photoelectrochemistry [14] and colorimetric assay [15] are developed to detect the activity of DNA MTase. Owing to the advantages of rapidity, simplicity, cost-effectiveness, and especially easy to read out by the naked eye, colorimetric assay has received an intensive attention. For example, Jiang's group [16] fabricated a colorimetric biosensor for DNA MTase detection based on MTase-protection of the DNA-gold nanoparticles (AuNPs). Li et al. [17] developed a novel strategy for detection of DNA MTase activity based on methylationresponsive DNAzyme with the detection limit of 6 U/mL. To amplify the signal, a label-free colorimetric method for DNA MTase assay based on methylation-blocked cascade amplification was established by Zhao et al. [18]. However, the instability of the AuNPs and the complex reaction conditions of enzymes may limit the use of the above methods. Therefore, a method that is more stable and simpler is still needed. Laterly, we have reported a novel electrochemical biosensors based on DNA functionalized nano mesoporous silica (MSNs) for assay of the DNA MTase activity [19]. Inspired by the unique feature of MSNs, such as biocompatibility, tunable pore structure, high thermal stability and large surface area, and the catalytic activity of Fe3O4, we developed a simple colorimetric approach for detection of DNA MTase activity based on the keypad lock of duplex DNA
modified meso-SiO2@Fe3O4. Owing to the report that the pore of the nano mesoporous silica can be blocked by a DNA duplex [20], the DNA MTase activity can be monitored by the color change of methylation-cleaved initiated catalytic reaction. Additionally, this way can be performed in human serum with satisfying recovery, and used for screening the antimicrobial drugs with high selectivity. 2. Experimental 2.1. Reagents and materials Tetraethylorthosilicate (TEOS), (3-aminopropyl) trimethoxysilane (APTES), 1-[3-(dimethylamino) propyl]-3ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosucnimide sodium salt (NHS), 3,30 ,5,50 -Tetramethylbenzidine (TMB), tris (hydroxymethyl) aminomethane (Tris), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were purchased from Aladdin (Shanghai, China). N-cetyltrimethylammonium bromide (CTAB) was obtained from Xinran Industrial Co., Ltd (Shanghai, China). Paclitaxel was purchased from Dibo Chemical Technology Co., Ltd. (Shanghai, China). FeSO4$7H2O, Fe2(SO4)3, H2O2, and other chemical reagents were all purchased from Kay Tong Chemical Reagents Co., Ltd (Tianjin, China). The serum samples were provided by the university hospital. All reagents employed were of analytical grade and the solutions were prepared by deioned water after high pressure steam sterilization. The Dam MTase, SAM and Dpn I endonuclease were supplied by New England Biolabs (Ipswich, MA) and Fermentas (MD, USA). According to the supplier, Dam MTase and restriction endonuclease Dpn I were stored at 20 C in the refrigerator in Dam MTase store buffer (50 mM KCl, 50 mM TriseHCl, 10 mM EDTA, 1 mM dithiothreitol, 200 mg/mL BSA and 50% glycerol, pH 7.5) and Dpn I store buffer (10 mM TriseHCl, 300 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 500 mg/mL BSA, and 50% Glycerol, pH 7.4), respectively. The DNA were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China) and stored in the TE buffer (10 mM TriseHCl, and 1 mM EDTA, pH 8.0) at 20 C in the refrigerator. The base sequences are as follows: DNA S1: 50 - COOH - ATAGT GATC ATTGTTATTAGGGAG - 30 DNA S2: 50 - CTCCCTAATAACAAT GATC ACTAT - 30 UVeVis absorption spectra was carried out on a UV-2450 Shimadzu Vis-spectrometer (Japan).Transmission electron microscopy (TEM) was performed on a JEM-100CX electron microscope (Japan). Fourier transform infrared (FTIR) spectra were obtained on a Thermo Nicolet-380 IR spectrophotometer (USA). 2.2. Synthesis of meso-SiO2@Fe3O4
Scheme 1. Schematic illustration of the Dam MTase activity colorimetric assay.
Briefly [21,22], a clear solution (150 mL) containing 5.6 mM
Fig. 1. TEM image of meso-SiO2@Fe3O4 (A) and (B). Photographs of meso-SiO2@Fe3O4 (C) and meso-SiO2@Fe3O4 attracted towards the magnet for 1 min (D).
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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FeSO4$7H2O and 11.2 mM Fe2(SO4)3 was heated to 50 C with the protection of N2, followed by the addition of 12.5 mL concentrated ammonia aqueous solution (NH3$H2O) under vigorous stirring. After 30 min, the black products were washed three times with water and dried at 60 C for 6 h. Then 0.1 g as-prepared Fe3O4 was mixed with 0.3 g CTAB, 1.0 g NH3$H2O, 80 mL deioned water and 60 mL ethanol, and stirred vigorously for 30 min. Subsequently, TEOS (0.4 g) was added dropwise to the mixture. After stirring for 2 h, the obtained products were collected with a magnet and washed with water and ethanol. In order to remove the template CTAB, the purified products were redispersed in 60 mL of methanol to refluxe for 48 h at 80 C. The extraction was repeated for three times, and the products were washed with ethanol. Finally, the products were redispersed in a mixture of 100 mL ethanol and 7 mL APTES. After ‘24 h’ stirring, the meso-SiO2@Fe3O4 were washed with deioned water and dried. FT-IR was carried out to confirm the existence of amine group on meso-SiO2@Fe3O4 (Fig. S1 in the Supporting Information). 2.3. Assembly of DNA DNA S1 (10 mL) was first activated by 50 mL of TriseHCl (pH 7.4) containing EDC (0.5 mg/mL) and NHS (0.5 mg/mL) for 2 h. Then 10 mL of meso-SiO2@Fe3O4 (4 g/L) was added and reacted for another 2 h. After magnetic separation, the meso-SiO2@Fe3O4-DNA S1 was washed three times with TE buffer and dispersed in 50 mL of solution containing 10 mM TriseHCl (pH 7.0), 1.0 mM EDTA and 1.0 M NaCl, followed the addition of 10 mL of DNA S2. Next, the mixture was incubated at 37 C for 2 h to form meso-SiO2@Fe3O4DNA S1-S2. Finally, the products were separated and washed, then saved in 10 mL of TE buffer. The UVevis spectroscopy was carried out to characterize that DNA have been assembled to mesoSiO2@Fe3O4 (Fig. S2). 2.4. Colorimetric assay of Dam MTase activity In a typical experiment, the methylation and cleavage was carried out by adding various concentration of Dam MTase, 80 mM
Fig. 2. UVeVis absorption spectra of different sample. (a) TMB and H2O2, (b) mesoSiO2@Fe3O4, TMB and H2O2, (c) meso-SiO2@Fe3O4-DNA S1, TMB and H2O2, (d) mesoSiO2@Fe3O4-DNA S1-S2, TMB and H2O2, (e) meso-SiO2@Fe3O4-DNA S1-S2, Dpn I, TMB and H2O2, (f) meso-SiO2@Fe3O4-DNA S1-S2, Dam MTase, Dpn I, TMB and H2O2. The corresponding photograph was also presented, respectively.
Fig. 3. (A) The relative UVeVis absorbance intensities catalyzed by meso-SiO2@Fe3O4 fixed with different concentration of DNA. (B) The relative UVeVis absorbance intensities to various catalytic reaction time. (C) The relative UVeVis absorbance intensities to different time of methylation reaction and cleaving. The Dam MTase concentration in all figures is 40 U/mL. Error bars show the standard deviation of three experiments.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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SAM, 1 Dam buffer (50 mM TriseHCl, 10 mM EDTA, 5 mM 2mercaptoethanol, pH 7.5), 50 U/mL Dpn I and 1 NEB buffer (20 mM Tris-acetate, 50 mM Potassium acetate, 10 mM magnesium acetate and 100 mg/mL BSA, pH 7.9) to 10 mL of TE buffer containing SiO2@Fe3O4-DNA S1-S2. The mixture was incubated at 37 C for 2 h. Then a magnet was used to collect the sediment. After washing with vast PBS buffer (0.1 M, pH 7.0), 90 mL of 0.1 M PBS buffer (pH 5.0), 20 mL of TMB (8 mM) and 40 mL of H2O2 (100 mM) was added successively. The absorption spectra were recorded in 20 min by UVeVis spectrometer. 2.5. Influence of paclitaxel on Dam MTase activity To investigate the effect of paclitaxel on Dam MTase activity, the similar procedures were performed with mentioned above, except adding of different concentrations of the inhibitor together with Dam MTase. 3. Results and discussion
color change from colorless to blue. At higher activity of Dam MTase, more DNA hybrid was methylated and cleaved, and more Fe3O4 were exposed, and thus, a stronger color change was obtained. In this way, by the naked eye or with simple UVeVis spectroscopy, we could detect the activity of Dam MTase quantitatively. 3.2. Characterization of meso-SiO2@Fe3O4 Fig. 1A depicted the TEM image of the meso-SiO2@Fe3O4 nanosphere with average diameter of 400 nm. It could be observed that the Fe3O4 core was well coated by a silica shell with thickness of about 30 nm. Moreover, abundant of mesopores in the silica shell could be obtained from HRTEM image (Fig. 1B). It demonstrated that the meso-SiO2@Fe3O4 have been successfully synthesized, which was consistent with previous work [25]. Additionally, the magnetic separability of meso-SiO2@Fe3O4 was supported by the experimental observation that the nanospheres in solution were attracted towards the magnet within 1 min (Fig. 1D), which could be redispersed again like Fig. 1C.
3.1. Principle of the colorimetric assay 3.3. The feasibility of the designed system The developed colorimetric strategy in profiling the MTase activity is described in Scheme 1. Primarily, the synthesized mesoSiO2@Fe3O4 was first assembled with DNA S1 through the reaction between the amino-group and carboxyl. After hybridization, the DNA duplex can serve as a cap to block pores of the meso-SiO2@Fe3O4 to prevent the contact of guest molecules with Fe3O4. Then a weak color change could be generated. Subsequently, in the presence of Dam MTase and SAM, the DNA hybrid was methylated. Since Dpn I is a methylation-sensitive restriction endonuclease, the methylated DNA hybrid can be cleaved at a specific site of 50 GAmTC e 30 [23] and formed a new hybrid. However, the new hybrid is unstable [24] and easy to separate into a single-stranded DNA fragment. Therefore, the lock is open and the colorimetric reaction of TMB and H2O2 is preformed by Fe3O4, who has been proved to be an excellent mimetic enzyme, resulting a significant
To demonstrate the feasibility of the designed system, several experiments were carried out. As shown in Fig. 2, characteristic absorption peaks at 652 nm for the oxidized TMB product could be observed. Because colorless TMB can be oxidized by H2O2 to yield a blue colored product, which can be recorded by the absorption spectra. To test the peroxidase-like activity of meso-SiO2@Fe3O4, catalytic oxidation of TMB in the presence of H2O2 was performed. Compare to the blank sample (curve a), the peak (curve b) increased significantly due to the catalytic of Fe3O4. When meso-SiO2@Fe3O4 first connected with DNA S1, the peak (curve c) remained. However, the absorbance (curve d) decreased after DNA hybridization, which proved that the DNA duplex can block pores of the meso-SiO2@Fe3O4 to prevent the contact of guest molecules with Fe3O4. Moreover, in the presence of Dam MTase and Dpn I, the significant
Fig. 4. (A) Photograph and (B) absorption spectra in the presence of different concentrations of Dam MTase. (C) Linear relationship between the absorption intensity and the Dam MTase concentration.
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028
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performance was the reaction time. In this method, a high colorimetric signal was expected to be achieved by a long time of catalytic reaction. The investigated results indicated that the absorbance intensity increased with the increase of catalytic reaction time (Fig. 3B). And 20 min was chosen for the following experiments due to the highest signal. Then, the time of methylation reaction and cleaving catalyzed by Dam MTase and Dpn I was optimized and the result was given in Fig. 3C. 120 min was selected because a plateau effect was reached after this time. 3.5. Methylation assay
Fig. 5. Selectivity of the designed strategy. Both of the concentrations of Dam MTase and M.SssI were 40 U/mL.
enhancement (curve f) showed the successful methylation and cleave of the hybridized DNA to form a new single-stranded DNA. On the contrary, without Dam MTase in the system, curve e was the same as curve d, indicating the unmethylated hybrid DNA cannot be cleaved by Dpn I. Besides, these results can also be estimated by the change of the color from colorless to various kinds of blue. Therefore, the sensitive characterization of Dam MTase activity can be successfully performed by this colorimetric biosensor.
To investigate the analytical performance of the designed strategy, Dam MTase at various concentrations were performed under the optimal conditions. As seen in Fig. 4A (left to right), the color changed from colorless to blue with the increasing of the Dam MTase concentrations. The corresponding UVevis absorption spectra were shown in Fig. 4B. As the concentration of Dam MTase increased, the absorption intensity increased. This was consistent with the fact that the more DNA hybrid was methylated and cleaved, the more Fe3O4 were exposed, and thus, a stronger color change can be obtained. Additionally, a good linear curve was gained between the absorption intensity and concentration of Dam MTase from 0 to 40 U/mL (Fig. 4C). With a correlation coefficient of 0.998, the regression equation was A ¼ 0.0092 c (U/mL) þ 0.068. The limit of detection of Dam MTase was approximately 0.73 U/mL in terms of the rule of 3s/k, which was compared with previously reported assays (Table S1 in the Supporting Information). 3.6. Selectivity study
3.4. Optimization of assay system To achieve the best analytical performance, the assay conditions were first optimized. Fig. 3A showed the absorbance of oxidized TMB, which was catalyzed by meso-SiO2@Fe3O4 fixed with various concentration of DNA. The colorimetric signal was declined as the concentration of DNA increased, and it reached a minimum at 10 mM. As a result, 10 mM was selected as the optimum concentration. The other significant factor that influence the sensing
Fig. 6. Absorption intensities obtained from the testing of TriseHCl buffer (blue column) and human serum sample (red column) spiked with Dam MTase. The concentrations of Dam MTase were 0 (control), 1, 10 and 40 U/mL. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Another factor to assess the performance of the method is selectivity. In order to evaluated, M. SssI was selected as an interference enzyme. As can be seen from Fig. 5, a significant enhancement of absorption intensity was obtained in the presence of Dam MTase. On the contrary, no distinct was observed in the presence of M. SssI. Because the recognition of the interference enzyme was 50 - CG -30 sequence which was different from that of Dam MTase. These results indicated that this method can easily discriminate Dam MTase from M.SssI MTase. Therefore, the designed strategy for Dam MTase activity detection possessed a good selectivity.
Fig. 7. Inhibition efficiency of Dam MTase by paclitaxel.
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3.7. Dam MTase sensing in human serum A significant challenge for assay of the MTase activity is to achieve the detection in complex biological sample. In this method, we investigated the standardized human serum sample spiked with Dam MTase at three different concentrations of Dam MTase. The human serum samples were diluted in 1:10 ratio by 50 mM TriseHCl buffer. As can be seen in Fig. 6, the absorbance intensity from the diluted human serum sample was well consistent with that of from TriseHCl buffer. The recoveries were in the range of 90.2%e102.5% with a maximum RSD of 5.62%. These results indicated that the designed method held a great promise application in high concentrations of interfering species and further potential for the practicality in complex biological samples. 3.8. Assay of the inhibition of Dam MTase activity To further extend the potential application of the designed biosensor, paclitaxel, an anticancer and antimicrobial drug, was chosen as a model to investigate the influence of inhibitors on DNA methylation. As shown in Fig. 7, increasing the inhibitor concentration, the inhibition efficiency gradually increased. The paclitaxel was found to inhibit 67.1% Dam MTase activity. In addition, the IC50 value (the inhibitor concentration required to reduce enzyme activity by 50%) was considered to be about 170 mM, which is similar to the previous report [26]. These results indicated that paclitaxel could inhibit the Dam MTase activity strongly. So the method could be used to screen inhibitors of Dam MTase. 4. Conclusion In conclusion, we have successfully developed a novel colorimetric assay for Dam MTase activity, using the control of the opening or closing of the meso-SiO2@Fe3O4 pores by DNA, which have an impact on the catalytic of TMB and H2O2. As a result, the asproposed biosensor offered a highly sensitive method for detection of Dam MTase activity with a wide range and a low detection of 0.73 U/mL. Importantly, this way can be performed in human serum with satisfying recovery, and used for screening the antimicrobial drugs with high selectivity. By exchanging the corresponding DNA sequence, the method could be extended to other DNA MTase detection such as cancer-related DNA MTase family and the corresponding inhibitor screening. Therefore, this method was expected to be useful for further application in disease diagnosis and drug development with these attractive analytical characteristics. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 21375079, 21105056), the Project of Development of Science and Technology of Shandong Province, China (No. 2013GZX20109) and the Natural Science Foundation of Shandong province China (No. ZR2014BQ029). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2016.03.028. References
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[1] A. Jeltsch, R.Z. Jurkowska, T.P. Jurkowski, K. Liebert, P. Rathert, M. Schlickenrieder, Application of DNA methyltransferases in targeted DNA
Please cite this article in press as: P. Liu, et al., A colorimetric assay of DNA methyltransferase activity based on the keypad lock of duplex DNA modified meso-SiO2@Fe3O4, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.03.028