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Cite this: Analyst, 2014, 139, 2674
Received 15th November 2013 Accepted 11th March 2014
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A signal-on fluorescent assay for DNA methyltransferase activity using a methylationresistant endonuclease† Quang Huy Quachab and Bong Hyun Chung*ab
DOI: 10.1039/c3an02129g www.rsc.org/analyst
A simple, rapid, and signal-on fluorescent assay was developed for activity analysis of DNA methyltransferase and for screening of its inhibitors based on a methylation-resistant endonuclease and SYBR Green I.
DNA methylation, one of the most important epigenetic modications in both prokaryotes and eukaryotes, plays a crucial role in many biological processes, including gene silencing, DNA repair, cellular differentiation, X-chromosome inactivation, embryogenesis, and bacterial virulence regulation.1 The process is catalyzed by DNA methyltransferase (MTase), which covalently transfers a methyl group from S-adenosylmethionine (SAM) to an adenine or cytosine residue at specic palindromic DNA sequences.2 Numerous studies have indicated that an aberrant level of DNA MTase is closely associated with the pathogenesis of various diseases, particularly cancer, in mammals and the level of virulence in bacteria.3 Notably, DNA MTase has been regarded as a novel type of cancer biomarker and as a potential target in the development of antimicrobial drugs.4 Therefore, developing an assay for the quantication and activity analysis of DNA MTases is critical for both life science research and early cancer diagnosis. The traditional methods utilized for DNA MTase detection, based on gel electrophoresis,5 radioactive labeling,6 immune reaction,7 and high-performance liquid chromatography (HPLC),8 are well established. However, these methods have some drawbacks, including time-consuming procedures, the need for dangerous radioactive substrates, or costly antibodies. To circumvent these limitations, a number of non-radioactive and non-immunological approaches have been proposed. For
a
BioNano Heath Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O Box 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806, South Korea. E-mail: [email protected]; Fax: +82-42-879-8594; Tel: +82-42-860-4442
b
Nanobiotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, South Korea † Electronic supplementary information (ESI) available: Experimental section and supporting data (Fig. S1–S4). See DOI: 10.1039/c3an02129g
2674 | Analyst, 2014, 139, 2674–2677
example, a variety of electrochemical biosensors have been designed for the quantitative analysis of DNA MTase activity.9 In addition, some colorimetric methods based on gold nanoparticles have been developed to assay for DNA MTase activity with convenient visual detection.10 Despite such improvements in terms of sensitivity and other advantages, these techniques require multistep processes for DNA immobilization (or DNA conjugation) and lengthy assay times. Several other strategies, such as DNAzyme11 and chemiluminescence,12 have also been employed to analyze the activity of DNA MTase. Although these strategies have the advantages of real-time detection and highthroughput potential, they require intricately designed DNA probes. Recently, a methyl-binding domain protein13 and carbon nanoparticles14 have been applied to evaluate DNA MTase activity. Unfortunately, the lengthy assay time and complicated synthesis processes of carbon nanoparticles and Bi2S3 nanorods limit the practical application of these approaches. A signal-off assay based on SYBR Green has been developed to analyze DNA methylase activity using DNA hairpin probes.15 More importantly, nearly all of the reported assays are designed by combining certain DNA MTases with a corresponding methylation-specic endonuclease that specially recognizes and cleaves the methylated sites. However, the use of methylation-specic endonucleases for the detection of other DNA MTases that recognize different palindromic DNA sequences is limited. Methylation-resistant endonuclease has also been used to evaluate DNA MTase activity in bioluminescent assay, but this method requires a complicated and timeconsuming step of protein expression in vitro.16 Hence, it is necessary to develop a simple, rapid, and economic assay based on methylation-resistant cleavage to offer a general approach for investigating more DNA MTases. SYBR Green I (SG) is a well-known sensitive uorescent dye used for staining DNA, and the strong interaction of SG with dsDNA can be used to discriminate ssDNA and dsDNA structures.17 In a previous study, we successfully applied SG to evaluate telomerase activity.18 Herein, we report a very simple and rapid assay for the uorescent detection of DNA MTase activity
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with acceptable sensitivity. According to this strategy, DNA adenine methyltransferase (Dam MTase) and DpnII are chosen as DNA MTase and methylation-resistant endonuclease model enzymes, respectively. An overview of the newly proposed method for Dam MTase detection without employing any further enhancement mechanism is depicted in Scheme 1. The doublestranded DNA (dsDNA) used in this study contains three 50 GATC-30 palindromic sites that are recognized by both Dam MTase and DpnII. With sufficient Dam MTase catalytic activity, these palindromic sites are methylated and resistant to cleavage by DpnII, and the addition of a SG solution leads to a sharp increase in uorescence intensity due to the interaction between SG and the intact dsDNA. Conversely, in the absence of Dam MTase or in the presence of Dam MTase inhibitors, the DNA probes are unmethylated and cleaved by DpnII; owing to their low melting temperature, the cleaved products are unstable at the experimental temperature (37 C). Therefore, the sharp increase in uorescence intensity is not observed aer the addition of SG. Before exploring the sensitivity of this proposed assay, there are two main issues that should be addressed. One is the concentration of the DNA probe, and the other is the amount of DpnII sufficient to cleave all the unmethylated 50 -GATC-30 sites. We found that, at a concentration of the DNA probe, it was impossible to discriminate the differences in uorescence intensity of Dam MTase solutions above 10 U ml1 (data not shown). We speculate that the DNA probe used was insufficient for methylation by the excess of Dam MTase used. However, we also found that increasing the DNA concentration resulted in a high background signal of the solution aer treatment with DpnII because of the interaction between ssDNA and SG. Considering all these effects, a concentration of the DNA probe of 0.5 mM (nal concentration in the methylation reaction) was chosen. At this DNA concentration, the uorescence ratio F/F0 of completely methylated and unmethylated solutions was found to be approximately 8–9 (data not shown). The effect of the DpnII concentration was investigated using data, which indicated that 20 units of DpnII were sufficient for the cleavage of the DNA probe used (Fig. S1†). However, considering the reference of the reaction time, 40 units of DpnII were chosen as the optimal amount. At this concentration of DpnII, the cleavage reaction lasted 30 minutes (as shown in Fig. S2†). As SAM acts as the donor of the methyl group, the effect of its concentration was also investigated: with an increasing SAM concentration, the uorescence intensity
Scheme 1 Schematic illustration of the proposed fluorescence assay for the detection of Dam MTase activity.
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increased and tended to reach a plateau at 80 mM (Fig. S3†). Hence, 80 mM of SAM was used for the ensuing experiments. Under the optimized conditions, a series of Dam MTase solutions with concentrations ranging from 0.2 to 100 U ml1 were analyzed to explore the sensitivity of this newly proposed assay. As depicted in Fig. 1A, the uorescence intensities increased with the increase in Dam MTase concentrations. In addition, the lack of a band of 30 base pairs in lane 2 (the inset of Fig. 1A) suggests that the DNA probe was cleaved by DpnII. In contrast, a clear single band of 30 base pairs is observed in lane 1 and is similar to that in lane 3 (the DNA probe without treatment with Dam MTase and DpnII). These results conrm the strategy of this assay, which is based on the fact that Dam MTase can protect a DNA probe from DpnII cleavage. SG then intercalates into the intact dsDNA, resulting in a remarkable increase in uorescence intensity.
Fig. 1 (A) Changes in fluorescence intensity with various concentrations of Dam MTase after the addition of SG solution. The arrow from a to l represents Dam MTase concentrations from 0, 0.2, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, and 100 U ml1. The inset shows an image of the assay of Dam MTase by gel electrophoresis: ladder (L), the DNA probe with treatment with both Dam MTase and DpnII (1), the DNA probe with treatment with DpnII only (2), and the DNA probe without treatment with Dam MTase and DpnII (3). (B) The calibration curve of fluorescence intensity responses to different concentrations of Dam MTase. The inset shows the linear correlation between the fluorescence intensity and the logarithm of the Dam MTase concentration. The error bars represent the standard deviations of three repeated experiments.
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Table 1
Communication A comparison of the proposed method with other reported assays for the detection of Dam MTase activity
Detection method
Strategy
Detection limit (U ml1)
Linear range (U ml1)
Detection time (minutes)
Ref.
Colorimetric Electrochemical Electrochemical Fluorescence Electrochemical Bioluminescence Chemiluminescence Colorimetric Fluorescence Fluorescence Electrochemical Electrochemical Fluorescence
Unmodied Au nanorod-based Single wall carbon nanotube-based Methylene blue-modied DNA probe DNAzyme-based Intercalating activity of RuHex Methylation-resistant endonuclease Hairpin probe-based DNAzyme-based Hairpin-shaped 8-17 DNAzyme Carbon nanoparticle-based Terminal transferase-mediated extension AuNP amplication Methylation-resistant endonuclease
0.14 0.04 0.07 0.2 0.18 0.08 1.29 104 6 0.4 0.1 0.04 0.12 0.16
0.1–30 0.1–1 0.1–1 0.5–40 0.25–10 0.2–100 0.025–400 6–100 0.4–20 0.5–100 0.1–20 0.2–10 1–100
300 370 420 165 245 340 365 150 150 120 100 120 95
19c 9b 9e 11a 9a 16 12 20 11b 14 9d 9c This study
Fig. 1B shows the calibration curve of the evaluation of the activity of Dam MTase, with the inset revealing a good linear relationship between the uorescence intensity and the logarithm of the concentration of Dam MTase in the range of 1–100 U ml1. This linear relationship can be described as Y ¼ 197.2X + 113.13, with a correlation coefficient of 0.993, where Y and X are the uorescence intensity and logarithm of the concentration of Dam MTase, respectively. The detection limit was calculated to be 0.16 U ml1 (in terms of the rule of 3 times standard deviation over the blank response), which is acceptable in comparison to some previously reported strategies (Table 1). Although our assay is not the best in terms of sensitivity, it has the following advantages: it is very simple and rapid (Table 1), and it is economical because it does not require a uorescein-labeled DNA probe. Furthermore, there is no enhancement mechanism used in this proposed assay. More importantly, it is a signal-on assay, which is based on the activity of a methylation-resistant endonuclease rather than a methylation-specic endonuclease. Therefore, it can be easily extended to investigate the activity of other DNA MTases. Inhibitors of DNA MTases have a broad spectrum of application in cancer therapy and as antimicrobial agents because DNA methylation plays an important role in many biological processes in both prokaryotes and eukaryotes.1 Therefore, an inhibition experiment was performed using different drugs to further extend the potential application of this assay in the screening of Dam MTase inhibitors. It is worthwhile to note that DpnII is included in this assay in addition to Dam MTase. Hence, the effects of Dam MTase inhibitors on the activity of DpnII were investigated prior to the inhibition experiments. Although these drugs had almost no effect on DpnII activity (Fig. S4†), they did affect the activity of Dam MTase to different degrees (Fig. 2A). Gentamicin showed the strongest inhibition, whereas the opposite was true for ampicillin at the same concentration (5 mM), consistent with those for previously reported assays.14,19 These observed results could be due to the Dam MTase used in the present study originating from E. coli and the fact that gentamicin is a broad-spectrum antibiotic agent.
2676 | Analyst, 2014, 139, 2674–2677
The inuence of the concentration of 5-uorouracil, a commonly used anticancer drug, on the activity of Dam MTase was explored in more detail; the results are shown in Fig. 2B.
(A) Effect of different types of drugs, 5F (5-fluorouracil), A (ampicillin), G (gentamicin), M (mitomycin), and B (benzylpenicillin) on the activity of Dam MTase. (B) Effect of the concentration of 5-flurouracil on the activity of Dam MTase. The IC50 value was calculated to be 6.38 mM. The error bars represent the standard deviations of three repeated experiments. Fig. 2
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Aer the samples were treated with 5-uorouracil, their uorescence intensities were signicantly decreased in a dosedependent manner, with an estimated IC50 value of 6.38 mM. These results demonstrate that the proposed assay has a potential application in screening DNA MTase-targeted drugs, which might be used as antibiotics and cancer therapeutics. In summary, we developed a label-free assay to convert Dam MTase activity into uorescent signals using methylationresistant endonuclease and SG. The method is based on the fact that the DNA probe is methylated by Dam MTase and thus resistant to cleavage by DpnII and that SG can discriminate between ssDNA and dsDNA. Using this assay, 0.16 U ml1 Dam MTase can be easily detected without any further enhancement. In addition, the total time for this process is 95 minutes, with a very simple protocol, making it more convenient than other methods. Because it uses a methylation-resistant endonuclease rather than a methylation-specic endonuclease, this novel method can be extended to analyze the activity of other DNA MTases. Owing to its simplicity and rapid nature, this label-free assay could be utilized for the initial screening of DNA MTase inhibitors as anticancer and antimicrobial agents.
Analyst
5
6
7 8 9
10
Acknowledgements This work was supported by BioNano Health-Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (grant number HGUARD_2013M3A6B2078950) and the KRIBB Initiative Program, Republic of Korea.
11
Notes and references
13
1 (a) P. A. Jones and S. B. Baylin, Nat. Rev. Genet., 2002, 3, 415; (b) D. A. Low, N. J. Weyand and M. J. Mahan, Infect. Immun., 2001, 69, 7197; (c) M. G. Marinus and J. Casadesus, FEMS Microbiol. Rev., 2009, 33, 488; (d) M. M. Suzuki and A. Bird, Nat. Rev. Genet., 2008, 9, 465. 2 (a) N. Mashhoon, M. Carroll, C. Pruss, J. Eberhard, S. Ishikawa, R. A. Estabrook and N. Reich, J. Biol. Chem., 2004, 279, 52075; (b) A. Jeltsch, ChemBioChem, 2002, 3, 274. 3 (a) J. A. Tsou, J. A. Hagen, C. L. Carpenter and I. A. L. Offringa, Oncogene, 2002, 21, 5450; (b) K. D. Robertson, Nat. Rev. Genet., 2005, 6, 597; (c) D. S. Shames, J. D. Minna and A. F. Gazdar, Curr. Mol. Med., 2007, 7, 85; (d) P. M. Das and R. Singal, J. Clin. Oncol., 2004, 22, 4632. 4 (a) M. A. Dawson and T. Kouzarides, Cell, 2012, 150, 12; (b) D. C. Wahnon, V. K. Shier and S. J. Benkovic, J. Am. Chem.
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12
14 15 16 17 18 19
20
Soc., 2001, 123, 976; (c) B. Brueckner and F. Lyko, Trends Pharmacol. Sci., 2004, 25, 551. (a) D. Stach, O. J. Schmitz, S. Stilgenbauer, A. Benner, H. Dohner, M. Wiessler and F. Lyko, Nucleic Acids Res., 2003, 31, e2; (b) G. W. Rebeck and L. Samson, J. Bacteriol., 1991, 173, 2068. (a) S. Som and S. Friedman, J. Biol. Chem., 1991, 266, 2937; (b) A. Bergerat, W. Guschlbabuer and G. V. Fazakerley, Proc. Natl. Acad. Sci. U. S. A., 1991, 88, 6394. E. Boye, M. G. Marinus and A. L. Olesen, J. Bacteriol., 1992, 174, 1682. K. C. Kuo, R. A. McCune and C. W. Gehrke, Nucleic Acids Res., 1980, 8, 4763. (a) G. L. Wang, L. Y. Zhou, H. Q. Luo and N. B. Li, Anal. Chim. Acta, 2013, 768, 76; (b) Y. Wang, X. He, K. Wang, J. Su, Z. Chen, G. Yan and Y. Du, Biosens. Bioelectron., 2013, 41, 238; (c) X. He, J. Su, Y. Wang, K. Wang, X. Ni and Z. Chen, Biosens. Bioelectron., 2011, 28, 298; (d) H. Wu, S. Liu, J. Jiang, G. Shen and R. Yu, Chem. Commun., 2012, 48, 6280; (e) J. Su, X. He, Y. Wang, K. Wang, Z. Chen and G. Yan, Biosens. Bioelectron., 2012, 36, 123. (a) G. Song, C. Chen, J. Ren and X. Qu, ACS Nano, 2009, 3, 1183; (b) T. Liu, J. Zhao, D. Zhang and G. Li, Anal. Chem., 2010, 82, 229. (a) X. H. Zhao, L. Gong, X. B. Zhang, B. Yang, T. Fu, R. Hu, W. Tan and R. Yu, Anal. Chem., 2013, 85, 3614; (b) T. Tian, H. Xiao, Y. Long, X. Zhang, S. Wang, X. Zhou, S. Liu and X. Zhou, Chem. Commun., 2012, 48, 10031. Y. Zeng, J. Hu, Y. Long and C. Zhang, Anal. Chem., 2013, 85, 6143. H. Yin, B. Sun, Y. Zhou, M. Wang, Z. Xu, Z. Fu and S. Ai, Biosens. Bioelectron., 2014, 51, 103. X. Ouyang, J. Liu, J. Li and R. Yang, Chem. Commun., 2012, 48, 88. Y. Long and X. Zhou, Proc. SPIE, 2012, 8329, 83290C1. C. Jiang, C. Yan, C. Huang, J. Jiang and R. Yu, Anal. Biochem., 2012, 423, 224. H. Zipper, H. Brunner, J. Bernhagen and F. Vitzthum, Nucleic Acids Res., 2004, 32, e103. Q. H. Quach, J. Jung, H. Kim and B. H. Chung, Chem. Commun., 2013, 49, 6596. (a) F. Chen and Y. Zhao, Analyst, 2013, 138, 284; (b) Y. Zhao, F. Chen, Y. Wu, Y. Dong and C. Fan, Biosens. Bioelectron., 2013, 42, 56; (c) X. Ju. Zheng, J. D. Qiu, L. Zhang, Z. X. Wang and R. P. Liang, Chem. Commun., 2013, 49, 2546. W. Li, Z. Liu, H. Lin, Z. Nie, J. Chen, X. Xu and S. Yao, Anal. Chem., 2010, 82, 1935.
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Cite this: Analyst, 2014, 139, 2674
Received 15th November 2013 Accepted 11th March 2014
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A signal-on fluorescent assay for DNA methyltransferase activity using a methylationresistant endonuclease† Quang Huy Quachab and Bong Hyun Chung*ab
DOI: 10.1039/c3an02129g www.rsc.org/analyst
A simple, rapid, and signal-on fluorescent assay was developed for activity analysis of DNA methyltransferase and for screening of its inhibitors based on a methylation-resistant endonuclease and SYBR Green I.
DNA methylation, one of the most important epigenetic modications in both prokaryotes and eukaryotes, plays a crucial role in many biological processes, including gene silencing, DNA repair, cellular differentiation, X-chromosome inactivation, embryogenesis, and bacterial virulence regulation.1 The process is catalyzed by DNA methyltransferase (MTase), which covalently transfers a methyl group from S-adenosylmethionine (SAM) to an adenine or cytosine residue at specic palindromic DNA sequences.2 Numerous studies have indicated that an aberrant level of DNA MTase is closely associated with the pathogenesis of various diseases, particularly cancer, in mammals and the level of virulence in bacteria.3 Notably, DNA MTase has been regarded as a novel type of cancer biomarker and as a potential target in the development of antimicrobial drugs.4 Therefore, developing an assay for the quantication and activity analysis of DNA MTases is critical for both life science research and early cancer diagnosis. The traditional methods utilized for DNA MTase detection, based on gel electrophoresis,5 radioactive labeling,6 immune reaction,7 and high-performance liquid chromatography (HPLC),8 are well established. However, these methods have some drawbacks, including time-consuming procedures, the need for dangerous radioactive substrates, or costly antibodies. To circumvent these limitations, a number of non-radioactive and non-immunological approaches have been proposed. For
a
BioNano Heath Guard Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O Box 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806, South Korea. E-mail: [email protected]; Fax: +82-42-879-8594; Tel: +82-42-860-4442
b
Nanobiotechnology, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-333, South Korea † Electronic supplementary information (ESI) available: Experimental section and supporting data (Fig. S1–S4). See DOI: 10.1039/c3an02129g
2674 | Analyst, 2014, 139, 2674–2677
example, a variety of electrochemical biosensors have been designed for the quantitative analysis of DNA MTase activity.9 In addition, some colorimetric methods based on gold nanoparticles have been developed to assay for DNA MTase activity with convenient visual detection.10 Despite such improvements in terms of sensitivity and other advantages, these techniques require multistep processes for DNA immobilization (or DNA conjugation) and lengthy assay times. Several other strategies, such as DNAzyme11 and chemiluminescence,12 have also been employed to analyze the activity of DNA MTase. Although these strategies have the advantages of real-time detection and highthroughput potential, they require intricately designed DNA probes. Recently, a methyl-binding domain protein13 and carbon nanoparticles14 have been applied to evaluate DNA MTase activity. Unfortunately, the lengthy assay time and complicated synthesis processes of carbon nanoparticles and Bi2S3 nanorods limit the practical application of these approaches. A signal-off assay based on SYBR Green has been developed to analyze DNA methylase activity using DNA hairpin probes.15 More importantly, nearly all of the reported assays are designed by combining certain DNA MTases with a corresponding methylation-specic endonuclease that specially recognizes and cleaves the methylated sites. However, the use of methylation-specic endonucleases for the detection of other DNA MTases that recognize different palindromic DNA sequences is limited. Methylation-resistant endonuclease has also been used to evaluate DNA MTase activity in bioluminescent assay, but this method requires a complicated and timeconsuming step of protein expression in vitro.16 Hence, it is necessary to develop a simple, rapid, and economic assay based on methylation-resistant cleavage to offer a general approach for investigating more DNA MTases. SYBR Green I (SG) is a well-known sensitive uorescent dye used for staining DNA, and the strong interaction of SG with dsDNA can be used to discriminate ssDNA and dsDNA structures.17 In a previous study, we successfully applied SG to evaluate telomerase activity.18 Herein, we report a very simple and rapid assay for the uorescent detection of DNA MTase activity
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with acceptable sensitivity. According to this strategy, DNA adenine methyltransferase (Dam MTase) and DpnII are chosen as DNA MTase and methylation-resistant endonuclease model enzymes, respectively. An overview of the newly proposed method for Dam MTase detection without employing any further enhancement mechanism is depicted in Scheme 1. The doublestranded DNA (dsDNA) used in this study contains three 50 GATC-30 palindromic sites that are recognized by both Dam MTase and DpnII. With sufficient Dam MTase catalytic activity, these palindromic sites are methylated and resistant to cleavage by DpnII, and the addition of a SG solution leads to a sharp increase in uorescence intensity due to the interaction between SG and the intact dsDNA. Conversely, in the absence of Dam MTase or in the presence of Dam MTase inhibitors, the DNA probes are unmethylated and cleaved by DpnII; owing to their low melting temperature, the cleaved products are unstable at the experimental temperature (37 C). Therefore, the sharp increase in uorescence intensity is not observed aer the addition of SG. Before exploring the sensitivity of this proposed assay, there are two main issues that should be addressed. One is the concentration of the DNA probe, and the other is the amount of DpnII sufficient to cleave all the unmethylated 50 -GATC-30 sites. We found that, at a concentration of the DNA probe, it was impossible to discriminate the differences in uorescence intensity of Dam MTase solutions above 10 U ml1 (data not shown). We speculate that the DNA probe used was insufficient for methylation by the excess of Dam MTase used. However, we also found that increasing the DNA concentration resulted in a high background signal of the solution aer treatment with DpnII because of the interaction between ssDNA and SG. Considering all these effects, a concentration of the DNA probe of 0.5 mM (nal concentration in the methylation reaction) was chosen. At this DNA concentration, the uorescence ratio F/F0 of completely methylated and unmethylated solutions was found to be approximately 8–9 (data not shown). The effect of the DpnII concentration was investigated using data, which indicated that 20 units of DpnII were sufficient for the cleavage of the DNA probe used (Fig. S1†). However, considering the reference of the reaction time, 40 units of DpnII were chosen as the optimal amount. At this concentration of DpnII, the cleavage reaction lasted 30 minutes (as shown in Fig. S2†). As SAM acts as the donor of the methyl group, the effect of its concentration was also investigated: with an increasing SAM concentration, the uorescence intensity
Scheme 1 Schematic illustration of the proposed fluorescence assay for the detection of Dam MTase activity.
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Analyst
increased and tended to reach a plateau at 80 mM (Fig. S3†). Hence, 80 mM of SAM was used for the ensuing experiments. Under the optimized conditions, a series of Dam MTase solutions with concentrations ranging from 0.2 to 100 U ml1 were analyzed to explore the sensitivity of this newly proposed assay. As depicted in Fig. 1A, the uorescence intensities increased with the increase in Dam MTase concentrations. In addition, the lack of a band of 30 base pairs in lane 2 (the inset of Fig. 1A) suggests that the DNA probe was cleaved by DpnII. In contrast, a clear single band of 30 base pairs is observed in lane 1 and is similar to that in lane 3 (the DNA probe without treatment with Dam MTase and DpnII). These results conrm the strategy of this assay, which is based on the fact that Dam MTase can protect a DNA probe from DpnII cleavage. SG then intercalates into the intact dsDNA, resulting in a remarkable increase in uorescence intensity.
Fig. 1 (A) Changes in fluorescence intensity with various concentrations of Dam MTase after the addition of SG solution. The arrow from a to l represents Dam MTase concentrations from 0, 0.2, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, and 100 U ml1. The inset shows an image of the assay of Dam MTase by gel electrophoresis: ladder (L), the DNA probe with treatment with both Dam MTase and DpnII (1), the DNA probe with treatment with DpnII only (2), and the DNA probe without treatment with Dam MTase and DpnII (3). (B) The calibration curve of fluorescence intensity responses to different concentrations of Dam MTase. The inset shows the linear correlation between the fluorescence intensity and the logarithm of the Dam MTase concentration. The error bars represent the standard deviations of three repeated experiments.
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Table 1
Communication A comparison of the proposed method with other reported assays for the detection of Dam MTase activity
Detection method
Strategy
Detection limit (U ml1)
Linear range (U ml1)
Detection time (minutes)
Ref.
Colorimetric Electrochemical Electrochemical Fluorescence Electrochemical Bioluminescence Chemiluminescence Colorimetric Fluorescence Fluorescence Electrochemical Electrochemical Fluorescence
Unmodied Au nanorod-based Single wall carbon nanotube-based Methylene blue-modied DNA probe DNAzyme-based Intercalating activity of RuHex Methylation-resistant endonuclease Hairpin probe-based DNAzyme-based Hairpin-shaped 8-17 DNAzyme Carbon nanoparticle-based Terminal transferase-mediated extension AuNP amplication Methylation-resistant endonuclease
0.14 0.04 0.07 0.2 0.18 0.08 1.29 104 6 0.4 0.1 0.04 0.12 0.16
0.1–30 0.1–1 0.1–1 0.5–40 0.25–10 0.2–100 0.025–400 6–100 0.4–20 0.5–100 0.1–20 0.2–10 1–100
300 370 420 165 245 340 365 150 150 120 100 120 95
19c 9b 9e 11a 9a 16 12 20 11b 14 9d 9c This study
Fig. 1B shows the calibration curve of the evaluation of the activity of Dam MTase, with the inset revealing a good linear relationship between the uorescence intensity and the logarithm of the concentration of Dam MTase in the range of 1–100 U ml1. This linear relationship can be described as Y ¼ 197.2X + 113.13, with a correlation coefficient of 0.993, where Y and X are the uorescence intensity and logarithm of the concentration of Dam MTase, respectively. The detection limit was calculated to be 0.16 U ml1 (in terms of the rule of 3 times standard deviation over the blank response), which is acceptable in comparison to some previously reported strategies (Table 1). Although our assay is not the best in terms of sensitivity, it has the following advantages: it is very simple and rapid (Table 1), and it is economical because it does not require a uorescein-labeled DNA probe. Furthermore, there is no enhancement mechanism used in this proposed assay. More importantly, it is a signal-on assay, which is based on the activity of a methylation-resistant endonuclease rather than a methylation-specic endonuclease. Therefore, it can be easily extended to investigate the activity of other DNA MTases. Inhibitors of DNA MTases have a broad spectrum of application in cancer therapy and as antimicrobial agents because DNA methylation plays an important role in many biological processes in both prokaryotes and eukaryotes.1 Therefore, an inhibition experiment was performed using different drugs to further extend the potential application of this assay in the screening of Dam MTase inhibitors. It is worthwhile to note that DpnII is included in this assay in addition to Dam MTase. Hence, the effects of Dam MTase inhibitors on the activity of DpnII were investigated prior to the inhibition experiments. Although these drugs had almost no effect on DpnII activity (Fig. S4†), they did affect the activity of Dam MTase to different degrees (Fig. 2A). Gentamicin showed the strongest inhibition, whereas the opposite was true for ampicillin at the same concentration (5 mM), consistent with those for previously reported assays.14,19 These observed results could be due to the Dam MTase used in the present study originating from E. coli and the fact that gentamicin is a broad-spectrum antibiotic agent.
2676 | Analyst, 2014, 139, 2674–2677
The inuence of the concentration of 5-uorouracil, a commonly used anticancer drug, on the activity of Dam MTase was explored in more detail; the results are shown in Fig. 2B.
(A) Effect of different types of drugs, 5F (5-fluorouracil), A (ampicillin), G (gentamicin), M (mitomycin), and B (benzylpenicillin) on the activity of Dam MTase. (B) Effect of the concentration of 5-flurouracil on the activity of Dam MTase. The IC50 value was calculated to be 6.38 mM. The error bars represent the standard deviations of three repeated experiments. Fig. 2
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Aer the samples were treated with 5-uorouracil, their uorescence intensities were signicantly decreased in a dosedependent manner, with an estimated IC50 value of 6.38 mM. These results demonstrate that the proposed assay has a potential application in screening DNA MTase-targeted drugs, which might be used as antibiotics and cancer therapeutics. In summary, we developed a label-free assay to convert Dam MTase activity into uorescent signals using methylationresistant endonuclease and SG. The method is based on the fact that the DNA probe is methylated by Dam MTase and thus resistant to cleavage by DpnII and that SG can discriminate between ssDNA and dsDNA. Using this assay, 0.16 U ml1 Dam MTase can be easily detected without any further enhancement. In addition, the total time for this process is 95 minutes, with a very simple protocol, making it more convenient than other methods. Because it uses a methylation-resistant endonuclease rather than a methylation-specic endonuclease, this novel method can be extended to analyze the activity of other DNA MTases. Owing to its simplicity and rapid nature, this label-free assay could be utilized for the initial screening of DNA MTase inhibitors as anticancer and antimicrobial agents.
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Acknowledgements This work was supported by BioNano Health-Guard Research Center funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea as Global Frontier Project (grant number HGUARD_2013M3A6B2078950) and the KRIBB Initiative Program, Republic of Korea.
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