Biosensors and Bioelectronics 81 (2016) 111–116
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Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy Ying Li, Chuanfeng Yu, Huixia Han, Caisheng Zhao, Xiaoru Zhang n Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 27 November 2015 Received in revised form 19 February 2016 Accepted 23 February 2016 Available online 24 February 2016
A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57 10 4 U mL 1. This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. & 2016 Elsevier B.V. All rights reserved.
Keywords: Surface-enhanced Raman scattering Target triggering primer generation DNA methyltransferase Multiple signal amplification
1. Introduction DNA methylation is a common gene protection approach, which is carried out by the catalysis of DNA methyltransferase (MTase) and plays an important role in both prokaryotes and eukaryotes (Jones and Takai, 2001). In the process of DNA methylation, a methyl group can be transferred from a donor molecule (e.g., S-adenosyl-L-methionine (SAM)) to the target cytosine or adenine in the specific DNA palindromic sequences (Cheng and Roberts, 2001; Onyango et al., 2002; Smith et al., 2012; Heyn et al., 2012). It is reported that DNA methylation plays crucial roles in DNA replication and repair, the regulation of gene transcription, genome imprinting and X-chromosome inactivation (Wei et al., 2014; Chen and Riggs, 2011; Chuang et al., 2012; Bernstein et al., 2007). Aberrant DNA methylation can be regarded as a key biomarker of genetic disease, embryonic development and cancer progression (Williams et al., 2011; Hill et al., 2011; Li et al., 2007; Wang et al., 2010). Hence, detection of DNA methylation and assay of MTase activity have received more and more research interests in recent years. Conventional methods for detecting DNA MTase include methylation-specific polymerase chain reaction (PCR) (Herman et al., 1996; Yang et al., 2004), high-performance liquid chromatography n
Corresponding author. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.bios.2016.02.057 0956-5663/& 2016 Elsevier B.V. All rights reserved.
(HPLC) (Friso et al., 2002; Reenila et al., 1995), gel electrophoresis (Rebeck and Samson, 1991; Xiong and Laird, 1997), and radioactive labeling (Som and Friedman, 1991; Nephew et al., 2009; Bergerat et al., 1991). Unfortunately, most of these methods are time-consuming with laborious operations or require radiolabeled substrates. For avoiding the above shortcomings, some alternative approaches are developed (Song et al., 2009; Li et al., 2012; Wu et al., 2012; Wang et al., 2006). In particular, isothermal cycle amplification is used in some of these methods for improving the sensitivity of MTase detection. For example, Zhang and his coworkers developed a rolling circle amplification (RCA)-based chemiluminescence method combining Nicking enzyme with Vent (exo-) polymerase (Zeng et al., 2013). Zhu et al. developed a method of hybridization chain reaction-based branched rolling circle amplification for chemiluminescence detection of DNA MTase (Bi et al., 2013). Yuan and his co-workers proposed a fluorescence method for the assay of MTase activity based on exonuclease-mediated target recycling (Xing et al., 2014). As an excellent detection method, surface enhanced Raman scattering (SERS) is now under active investigation (Nie and Emory, 1997; Stiles et al., 2008) due to its unique advantages as follows: (i) high sensitivity and specificity; (ii) “fingerprinting” ability and resistance of photobleaching; (iii) nondestructive, noninvasive for biological samples and ease of operation. Up to now, a few isothermal cycle amplification methods have been developed for SERS detection of DNA (Hu and Zhang, 2010), protein (Gao et al., 2015) and small molecules (Li et al., 2014; Ye et al., 2013).
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However, it has rarely been used for the assay of DNA MTase activity. In this study, we employ for the first time a target triggering primer generation-based multiple signal amplification strategy for detecting DNA MTase coupled with surface-enhanced Raman scattering (SERS) technique. In comparison with the conventional MTase assay methods, this strategy is unique in some characteristics: (i) By using of a duplex substrate, two hairpin templates and a Raman probe, multiple-signal amplification mode is achieved. A wide dynamic range and remarkably high sensitivity can be obtained under isothermal conditions. (ii) As the ‘primers’ can be generated successively during the DNA methylation reaction, the proposed method is being free from troublesome design and usage of exogenous primers. (iii) This novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase.
2. Experimental 2.1. Materials and apparatus Dam Methytransferase, Dpn I endonuclease, S-adenosylmethionine (SAM), Nb.BbvCI endonuclease and the corresponding buffer solution were purchased from New England Biolabs Inc. (Beverly, MA, USA) Klenow fragment of Escherichia coli DNA polymerase was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The mixture of four dNTPs (10 mM for each component) was purchased from SBS Genetech Co., Ltd. (Beijing, China). All oligonucleotides used in the present study were synthesized and purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China), and the sequences were listed in Table S1 (Supporting information). Hydrogen tetrachloroaurate(III) tetrhydrate (HAuCl4 4H2O) and trisodium citrate were ordered from Sigma-Aldrich. Other chemicals employed were of analytical reagent grade and were used without further purification. Doubly distilled water was used throughout the experiments. Magnetic beads coated with carboxyl groups (MBs–COOH) were purchased from Tianjin BaseLine ChroTech Research Center (Tianjin, China) and the gold chip used for Raman detection was purchased from BioNavis Ltd (Finland). Transmission electron microscopy (TEM) image was taken with JEM-2100 instrument (JEOL, Japan). UV–vis absorption spectra were obtained with a Cary 50 UV–vis–NIR spectrophotometer (Varian, Australia). Fluorescence measurements were carried out at a Hitachi F-4600 fluorometer (Hitachi Ltd., Japan). Raman measurements were performed on an inVia Raman Microscope (Renishaw, England). A microscope equipped with a 50 objective was used to focus the incident excitation laser and the excitation laser is 633 nm. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was carried out on the Tanon EPS-300 power supply (Tanon Science & Technology Co., Ltd., Shanghai, China), and the PAGE patterns were imaged on a WD-9413B gel imaging system (Beijing Liuyi Instrument Factory, Beijing, China). 2.2. Preparation of the Raman probe The AuNPs (gold nanoparticles)-functionalized Raman probe was obtained by capping the capture DNA (S3) and Barcode DNA (S4) on the surface of AuNPs according to the literature (Li et al., 2011). Briefly, 10 mL of 1.0 10 7 M capture DNA (S3, 5′-thiol) and 50 mL of 1.0 10 6 M signal DNA (S4, 5′-thiol and 3′-ROX) were mixed together and added to freshly prepared 1 mL AuNPs. Then the solution was shaken gently overnight at 37 °C. After that, the DNA/AuNPs conjugates were aged in salts (0.05 M NaCl for 6 h and
0.01 M NaCl for 6 h, respectively). The excess reagents were removed by centrifuging for 30 min at 10,000 rpm, and the obtained red precipitates were washed and centrifuged repeatedly for three times. The resulting Raman signal probes were dispersed into a phosphate buffer solution (PBS, 0.01 M, pH 7.4) and stored at 4 °C for further use. 2.3. Immobilization of Hairpin-probe (H2) on the surface of MBs Immobilization of Hairpin-probe (H2) on the surface of MBs was performed according to the reference with a slight modification (Zhang et al., 2009). 5 μL of carboxylated MBs was washed with imidazol-HCl buffer (pH 6.8, 0.1 M, 200 μL) for three times before use and then activated in 100 μL of 0.1 M EDC (imidazolHCl buffer, pH 6.8) at 37 °C for 30 min. After washing three times with 200 mL of 0.01 M PBS buffer (pH 7.4), 20 μL (10 6 M) of amino group modification Hairpin-probe (H2) was added to the MBs and incubated at 37 °C overnight. Finally, the excess Hairpin-probe was removed by magnetic separation and the resulting MBs-H2 was washed with PBS for three times, resuspended in 5 mL of PBS, and stored at 4 °C for further use. 2.4. Analysis of Dam MTase The duplex substrate of Dam MTase was prepared by mixing S1 (2.5 μL, 10 5 M) and S2 (2.5 μL, 10 5 M) at 37 °C for 0.5 h followed by the methylation process, which was carried out in 10 μL of reaction mixture containing the duplex substrate, various amounts of Dam MTase, 1 μL of 10 Dam Methyltransferase reaction buffer, 160 μM SAM, 1 μL of 10 CutSmart buffer and 20 U Dpn I. The experiment was performed at 37 °C for 1 h and the mixture was heated at 80 °C for 20 min for terminating the methylation reaction. Then the product was added to another 30 μL of reaction mixture containing the hairpin template (H1), MBs-H2, 6 μL Raman probe, 4 μL of 10 Klenow buffer, 0.20 U μL 1 of Klenow polymerase, 4 μL of 10 CutSmart buffer, 0.3 U μL 1 of Nb.BbvCI and 2 μL dNTPs. The experiment was performed at 37 °C for 1.5 h and the reaction was terminated by heating the mixture at 80 °C for 20 min to inactivate the enzymes. 2.5. Inhibition assay The inhibition of the drugs (gentamycin, benzylpenicillin, 5-fluorouracil, and mitomycin) on the activity of Dam MTase was investigated in the following experiment. First, 1 μM disparate drugs were separately mixed with the duplex substrate, 8 U/mL of Dam MTase, 1 μL of 10 Dam buffer, 160 μM SAM, after incubating the mixture for 2 h at 37 °C, 4 μL of 10 CutSmart buffer and 20 U Dpn I was added. The following procedures were the same as the analysis of Dam MTase. The effect of these inhibitors on the other enzymes including Dpn I, Klenow polymerase and NEase was shown in Supporting information. 2.6. Raman measurement The MBs incorporated with Raman probes were washed with PBS for three times to remove the additional reagents and redispersed in 15 μL of 0.01 M PBS (pH 7.4). Then 1.5 μL of each suspension was casted onto the gold chip and air-dried at room temperature before Raman analysis. The Raman spectra were measured by a Renisaw inVia Raman spectrometer at an excitation laser of 633 nm. The laser power was 5 mW, and the acquisition time for each spectrum was 5 s. Three repeated experiments were performed, the spectra were obtained from three different cites of each sample and calibrated with the WiRE Raman Software Version 3.3.
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2.7. Nondenaturing polyacrylamide gel electrophoresis (PAGE) The species produced in the process of DNA methylation and the strand-displacement amplification (SDA) were characterized by nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis (Acr¼acrylamide, Bis ¼N,N′-methylenebisacrylamide; Acr/Bis¼29/1). Tris–acetate-EDTA (TAE) (pH ¼8.5) was used as the separation buffer and the PAGE was generally run at 120 V for 1.5 h with loading of 10 μL of each sample into the lanes.
3. Results and discussion 3.1. The design principle of the SERS method for DNA MTase assay As models, DNA adenine methylation (Dam) MTase and Dpn I endonuclease are used in our experiments, both having the same special recognition sequence of the symmetric tetranucleotide 5′G-A-T-C-3′. Scheme 1 depicts the principle for analyzing the activity of Dam MTase. The duplex substrate is formed by the hybridization of DNA strands (S1 and S2). In the presence of Dam MTase, the symmetric 5′-G-A-T-C-3′of the substrate can be recognized and Dam MTase catalyzes the transfer of a methyl group from SAM to the N6 position of the adenine residues. Then, the methylated duplex substrate is cleaved into two parts by the methylation-sensitive restriction endonuclease Dpn I. In each part, there are only 6-complementary bases, so they are rapidly changed into two longer single-stranded DNAs (ssDNA, 12-base) and two shorter ssDNAs (6-base). Then the 12-base ssDNAs ingeniously functions as the primers (P1 and P2 in Scheme 1) of the strand-displacement amplification (SDA), which is achieved with
Scheme 1. Schematic Illustration of the proposed TPG-EXPAR strategy for Dam MTase activity assay.
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two repeated cycles of polymerization, nicking and displacement reactions, each is assisted by one primer. Once the primer (P1 or P2) hybridizes with the hairpin template (H1), it can extend along the template with the assistant of Klenow fragment polymerase and form a double-stranded DNA (dsDNA) containing the recognition site of nicking endonuclease (NEase). Subsequent singlestranded nicking produces a new replication site for polymerase and new ssDNAs (P3) in large quantities are released. Then P3 hybridizes with the loop part of hairpin template (H2) immobilized on the surface of magnetic beads (MBs–COOH). With the extension of P3, the hairpin structure of H2 is opened and the capture DNA (S3) conjugated on the Raman probe is attached to the opened stem and functions as a primer to trigger a new strand-displacement amplification. In the process of primer extension, a large number of Raman probes can be anchored on the surface of MBs by forming double-stranded DNAs and used for SERS measurement. 3.2. Feasibility of the assay The feasibility of our method for the assay of Dam MTase activity was evaluated by conducting a series of control experiments and the detection was based on the SERS signals of the Rox reporter on the Raman probes. In the absence of either Dam MTase or Dpn I, the duplex substrate could not be cleaved and no primer was produced for cycle amplification reaction, so the Raman signals were hardly observed (Fig. 1, curve a and b). Similarly, in the absence of either polymerase or NEase, SDA could not be performed and no Raman probes could be anchored on the surface of MBs, so the Raman signals were also very low only due to the nonspecific adsorption (Fig. 1, curve c and d). In contrast, in the presence of Dam MTase, Dpn I, polymerase and NEase, the Raman intensity was greatly raised (Fig. 1, curve e), which confirmed the feasibility of the proposed method for the assay of Dam MTase activity. For further confirming the feasibility of the method, we performed the experiment of nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis. The variation of DNA in the processes of DNA methylation and the strand-displacement amplification (SDA) was shown in Fig. 2. Compared to lane 1 (the duplex of S1 and S2), there was a new band appeared in lane 2 when both Dam MTase and Dpn I were added to the test solution of S1 and S2, indicating that a methylation reaction has taken place and the methylated DNA was then cut into small pieces. The product (P1
Fig. 1. SERS spectra obtained by a series of control experiments: in the presence of Dpn I, polymerase and NEase (a), Dam, polymerase and NEase (b), Dam, Dpn I and NEase (c), Dam, Dpn I and polymerase (d), Dam, Dpn I, polymerase and NEase (e).
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Fig. 2. Polyacrylamide gel electrophoresis (PAGE) of the DNA ladder marker (M); the duplex substrate (S1 and S2) (1); the duplex substrate treated with Dam and Dpn I (2); the SDA product of the hairpin probe (H1) and P1, P2 (the product of methylation reaction); the hairpin probe, H1 (4).
and P2 in Scheme 1) of methylation reaction was then used to demonstrate the validity of strand-displacement amplification (SDA). In lane 4, there was an obvious migration band corresponded to the hairpin template (H1). After mixed with P1, P2 (the product of methylation reaction), NEase, polymerase and dNTPs, following by incubating the mixture at 37 °C for 90 min, the product was used for PAGE and the result was shown in lane 3 (Fig. 2). It could be seen that two new migration bands appeared, which indicated that SDA was performed very well. 3.3. Analytical performance For investigating the analytical performance of this strategy, different concentrations of Dam MTase were determined under the optimal experimental conditions. Fig. 3A showed the variance of Raman scattering intensity with the concentration of Dam MTase. It could be seen that the representative vibrational peaks of Rox (1499 cm 1 and 1644 cm 1) were displayed and the corresponding Raman intensity at 1499 cm 1 was utilized to quantitatively evaluate the Raman response to Dam MTase activity. As could be seen from Fig. 3B, the Raman intensity had a good linear fit to the logarithm of Dam MTase over a range of 4 orders of magnitude from 0.001 to 10 U/mL. The regression equation is ΔI ¼0.240 lgC þ0.762 (ΔI is the normalized Raman intensity subtracting the blank and C is the concentration of Dam MTase), and the correlation coefficient (R) is 0.992. A detection limit of 2.57 10 4 U mL 1 can be estimated using a S/N (signal/noise)
Fig. 3. (A) Raman spectra in response to different amounts of Dam MTase: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 1, 10 U mL 1 from curve a to h; (B) The calibration curve of relative Raman intensity versus the amount of Dam MTase. Error bars showed the standard deviation of three experiments.
ratio of 3. A series of eleven repetitive measurements of 0.01 U/mL Dam MTase were used for estimating the precision, and the relative standard deviation (RSD) was 5.9%, showing good reproducibility. The sensitivity of the proposed strategy has dramatically improved as compared with most of the other developed approaches and the comparison of different methods for Dam MTase activity detection is shown in Table S2 (Supporting information). The detection limit is 3 orders of magnitude lower than that of the hybridization chain reaction-based branched rolling circle amplification method (Bi et al., 2013), 2 orders of magnitude lower than that of the exonuclease-mediated target recycling based fluorescence detection (Xing et al., 2014) and comparable with that of hairpin probe-based primer generation rolling circle amplification method (Zeng et al., 2013). The high sensitivity could be attributed to the following factors. First, three strand-displacement amplification modes are existed in this method, making a large number of Raman probes be anchored on the surface of MBs for SERS detection. Second, AuNPs are used for preparing the Raman probes, which can not only load more Rox-DNA but also enhance the SERS signal. Third, magnetic separation can easily circumvent the high background by removing the excess regents.
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Fig. 4. Selectivity of the proposed method. The concentration of Dam MTase is 10 U mL 1, and the concentration of M.SssI and HhaI MTase is 50 U mL 1.
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Fig. 5. (A) Influence of different drugs on the activity of Dam MTase. (1) No drug, (2) gentamycin, (3) 5-fluorouracil, (4) benzylpenicillin, (5) mitomycin.
3.4. Specificity of the method for dam MTase detection Furthermore, we investigated the specificity of this assay for Dam MTase. In this respect, M.Sss I and HhaI MTase were introduced as interference methyltranferases, which methylates all cytosine residues within the double-stranded dinucleotide recognition sequence 5′-C-G-3′ and the internal cytosine residue of the sequence 5′-G-C-G-C-3′, respectively. As shown in Fig. 4, a significantly higher Raman signal was observed in the presence of Dam MTase, while M.Sss I and HhaI have no obvious Raman enhancement compared with that of the blank test, even when adding 5-fold more of Dam MTase. These results demonstrated that the proposed strategy exhibited high selectivity due to the specific site recognition of Dam MTase toward the duplex substrate. 3.5. Inhibition assay Dam MTase plays an important role in the virulence of bacterial pathogens and DNA methylation is closely related to our health and disease, thus screening of inhibitors for DNA MTase receives more and more interest. In this research, for checking whether our sensitive method can be employed for high-throughput screen of Dam MTase inhibitors, some drugs (gentamycin, benzylpenicillin, 5-fluorouracil, and mitomycin) were investigated. From Fig. 5, it could be seen that gentamycin, 5-fluorouracil and benzylpenicillin could distinctly inhibit the Dam MTase activity and gentamycin was the most serious one with the inhibition ratio of about 45%. However, mitomycin had almost no effect on the methylation. These results indicate that our method has the potential to screen the drugs as inhibitors of Dam MTase. On the other hand, besides Dam, Dpn I, Klenow polymerase and NEase (Nb.BbvCI) are involved in our assay system, so it is necessary to examine the effect of these inhibitors on the other enzymes. The control experiments and the corresponding results were shown in Supporting information (Figs. S7 and S8), which indicated that no obvious influence could be observed when the concentration of the drugs was 1 mM.
were spiked into PBS and normal human serum (20%) respectively. The recoveries obtained by comparing the measured amounts to that of added Dam MTase were found to vary from 93.4% to 100.8%. (see Supporting information, Table S3) Three replicate determinations at different concentration levels exhibited RSDs ranging from 5.2% to 6.9% (n ¼3). The results showed an acceptable coherence between the data obtained in diluted serum and those in PBS, confirming the potentiality of this sensing system for MTase detection in real samples with other potentially competing species coexisting.
4. Conclusions In summary, a novel SERS method of DNA MTase assay has been developed by using of a target triggering primer generationmultiple signal amplification reaction. Taking advantages of the specific recognition of Dam MTase and DpnI, high amplification efficiency of the strategy and easy operation of SERS detection, a wide dynamic range and remarkably high sensitivity are obtained. Compared with previously reported methods, the detection limit of 2.57 10 4 U mL 1 is very low and other methyltranferases can be easily discriminated from Dam MTase with high selectivity. In addition, this sensitive assay method is successfully applied in the screening of inhibitors for Dam MTase. Given the attractive analytical characteristics, this sensing strategy might hold great promise for DNA methylation related clinical diagnosis and other biomedical research.
Acknowledgment This work was financially supported by the National Natural Science Foundation of China (21305072, 21275003).
3.6. Determination of dam MTase in real sample Appendix A. Supplementary material For testing the availability of this proposed method in real samples, the performance of Dam MTase in a human real serum sample was implemented. Different concentrations of Dam MTase
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.02.057.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Sensitive SERS detection of DNA methyltransferase by target triggering primer generation-based multiple signal amplification strategy Ying Li, Chuanfeng Yu, Huixia Han, Caisheng Zhao, Xiaoru Zhang n Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 27 November 2015 Received in revised form 19 February 2016 Accepted 23 February 2016 Available online 24 February 2016
A novel and sensitive surface-enhanced Raman scattering (SERS) method is proposed for the assay of DNA methyltransferase (MTase) activity and evaluation of inhibitors by developing a target triggering primer generation-based multiple signal amplification strategy. By using of a duplex substrate for Dam MTase, two hairpin templates and a Raman probe, multiple signal amplification mode is achieved. Once recognized by Dam MTase, the duplex substrate can be cleaved by Dpn I endonuclease and two primers are released for triggering the multiple signal amplification reaction. Consequently, a wide dynamic range and remarkably high sensitivity are obtained under isothermal conditions. The detection limit is 2.57 10 4 U mL 1. This assay exhibits an excellent selectivity and is successfully applied in the screening of inhibitors for Dam MTase. In addition, this novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase. & 2016 Elsevier B.V. All rights reserved.
Keywords: Surface-enhanced Raman scattering Target triggering primer generation DNA methyltransferase Multiple signal amplification
1. Introduction DNA methylation is a common gene protection approach, which is carried out by the catalysis of DNA methyltransferase (MTase) and plays an important role in both prokaryotes and eukaryotes (Jones and Takai, 2001). In the process of DNA methylation, a methyl group can be transferred from a donor molecule (e.g., S-adenosyl-L-methionine (SAM)) to the target cytosine or adenine in the specific DNA palindromic sequences (Cheng and Roberts, 2001; Onyango et al., 2002; Smith et al., 2012; Heyn et al., 2012). It is reported that DNA methylation plays crucial roles in DNA replication and repair, the regulation of gene transcription, genome imprinting and X-chromosome inactivation (Wei et al., 2014; Chen and Riggs, 2011; Chuang et al., 2012; Bernstein et al., 2007). Aberrant DNA methylation can be regarded as a key biomarker of genetic disease, embryonic development and cancer progression (Williams et al., 2011; Hill et al., 2011; Li et al., 2007; Wang et al., 2010). Hence, detection of DNA methylation and assay of MTase activity have received more and more research interests in recent years. Conventional methods for detecting DNA MTase include methylation-specific polymerase chain reaction (PCR) (Herman et al., 1996; Yang et al., 2004), high-performance liquid chromatography n
Corresponding author. E-mail address: [email protected] (X. Zhang).
http://dx.doi.org/10.1016/j.bios.2016.02.057 0956-5663/& 2016 Elsevier B.V. All rights reserved.
(HPLC) (Friso et al., 2002; Reenila et al., 1995), gel electrophoresis (Rebeck and Samson, 1991; Xiong and Laird, 1997), and radioactive labeling (Som and Friedman, 1991; Nephew et al., 2009; Bergerat et al., 1991). Unfortunately, most of these methods are time-consuming with laborious operations or require radiolabeled substrates. For avoiding the above shortcomings, some alternative approaches are developed (Song et al., 2009; Li et al., 2012; Wu et al., 2012; Wang et al., 2006). In particular, isothermal cycle amplification is used in some of these methods for improving the sensitivity of MTase detection. For example, Zhang and his coworkers developed a rolling circle amplification (RCA)-based chemiluminescence method combining Nicking enzyme with Vent (exo-) polymerase (Zeng et al., 2013). Zhu et al. developed a method of hybridization chain reaction-based branched rolling circle amplification for chemiluminescence detection of DNA MTase (Bi et al., 2013). Yuan and his co-workers proposed a fluorescence method for the assay of MTase activity based on exonuclease-mediated target recycling (Xing et al., 2014). As an excellent detection method, surface enhanced Raman scattering (SERS) is now under active investigation (Nie and Emory, 1997; Stiles et al., 2008) due to its unique advantages as follows: (i) high sensitivity and specificity; (ii) “fingerprinting” ability and resistance of photobleaching; (iii) nondestructive, noninvasive for biological samples and ease of operation. Up to now, a few isothermal cycle amplification methods have been developed for SERS detection of DNA (Hu and Zhang, 2010), protein (Gao et al., 2015) and small molecules (Li et al., 2014; Ye et al., 2013).
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However, it has rarely been used for the assay of DNA MTase activity. In this study, we employ for the first time a target triggering primer generation-based multiple signal amplification strategy for detecting DNA MTase coupled with surface-enhanced Raman scattering (SERS) technique. In comparison with the conventional MTase assay methods, this strategy is unique in some characteristics: (i) By using of a duplex substrate, two hairpin templates and a Raman probe, multiple-signal amplification mode is achieved. A wide dynamic range and remarkably high sensitivity can be obtained under isothermal conditions. (ii) As the ‘primers’ can be generated successively during the DNA methylation reaction, the proposed method is being free from troublesome design and usage of exogenous primers. (iii) This novel sensing system is potentially universal as the recognition element can be conveniently designed for other target analytes by changing the substrate of DNA MTase.
2. Experimental 2.1. Materials and apparatus Dam Methytransferase, Dpn I endonuclease, S-adenosylmethionine (SAM), Nb.BbvCI endonuclease and the corresponding buffer solution were purchased from New England Biolabs Inc. (Beverly, MA, USA) Klenow fragment of Escherichia coli DNA polymerase was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). The mixture of four dNTPs (10 mM for each component) was purchased from SBS Genetech Co., Ltd. (Beijing, China). All oligonucleotides used in the present study were synthesized and purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China), and the sequences were listed in Table S1 (Supporting information). Hydrogen tetrachloroaurate(III) tetrhydrate (HAuCl4 4H2O) and trisodium citrate were ordered from Sigma-Aldrich. Other chemicals employed were of analytical reagent grade and were used without further purification. Doubly distilled water was used throughout the experiments. Magnetic beads coated with carboxyl groups (MBs–COOH) were purchased from Tianjin BaseLine ChroTech Research Center (Tianjin, China) and the gold chip used for Raman detection was purchased from BioNavis Ltd (Finland). Transmission electron microscopy (TEM) image was taken with JEM-2100 instrument (JEOL, Japan). UV–vis absorption spectra were obtained with a Cary 50 UV–vis–NIR spectrophotometer (Varian, Australia). Fluorescence measurements were carried out at a Hitachi F-4600 fluorometer (Hitachi Ltd., Japan). Raman measurements were performed on an inVia Raman Microscope (Renishaw, England). A microscope equipped with a 50 objective was used to focus the incident excitation laser and the excitation laser is 633 nm. Nondenaturing polyacrylamide gel electrophoresis (PAGE) was carried out on the Tanon EPS-300 power supply (Tanon Science & Technology Co., Ltd., Shanghai, China), and the PAGE patterns were imaged on a WD-9413B gel imaging system (Beijing Liuyi Instrument Factory, Beijing, China). 2.2. Preparation of the Raman probe The AuNPs (gold nanoparticles)-functionalized Raman probe was obtained by capping the capture DNA (S3) and Barcode DNA (S4) on the surface of AuNPs according to the literature (Li et al., 2011). Briefly, 10 mL of 1.0 10 7 M capture DNA (S3, 5′-thiol) and 50 mL of 1.0 10 6 M signal DNA (S4, 5′-thiol and 3′-ROX) were mixed together and added to freshly prepared 1 mL AuNPs. Then the solution was shaken gently overnight at 37 °C. After that, the DNA/AuNPs conjugates were aged in salts (0.05 M NaCl for 6 h and
0.01 M NaCl for 6 h, respectively). The excess reagents were removed by centrifuging for 30 min at 10,000 rpm, and the obtained red precipitates were washed and centrifuged repeatedly for three times. The resulting Raman signal probes were dispersed into a phosphate buffer solution (PBS, 0.01 M, pH 7.4) and stored at 4 °C for further use. 2.3. Immobilization of Hairpin-probe (H2) on the surface of MBs Immobilization of Hairpin-probe (H2) on the surface of MBs was performed according to the reference with a slight modification (Zhang et al., 2009). 5 μL of carboxylated MBs was washed with imidazol-HCl buffer (pH 6.8, 0.1 M, 200 μL) for three times before use and then activated in 100 μL of 0.1 M EDC (imidazolHCl buffer, pH 6.8) at 37 °C for 30 min. After washing three times with 200 mL of 0.01 M PBS buffer (pH 7.4), 20 μL (10 6 M) of amino group modification Hairpin-probe (H2) was added to the MBs and incubated at 37 °C overnight. Finally, the excess Hairpin-probe was removed by magnetic separation and the resulting MBs-H2 was washed with PBS for three times, resuspended in 5 mL of PBS, and stored at 4 °C for further use. 2.4. Analysis of Dam MTase The duplex substrate of Dam MTase was prepared by mixing S1 (2.5 μL, 10 5 M) and S2 (2.5 μL, 10 5 M) at 37 °C for 0.5 h followed by the methylation process, which was carried out in 10 μL of reaction mixture containing the duplex substrate, various amounts of Dam MTase, 1 μL of 10 Dam Methyltransferase reaction buffer, 160 μM SAM, 1 μL of 10 CutSmart buffer and 20 U Dpn I. The experiment was performed at 37 °C for 1 h and the mixture was heated at 80 °C for 20 min for terminating the methylation reaction. Then the product was added to another 30 μL of reaction mixture containing the hairpin template (H1), MBs-H2, 6 μL Raman probe, 4 μL of 10 Klenow buffer, 0.20 U μL 1 of Klenow polymerase, 4 μL of 10 CutSmart buffer, 0.3 U μL 1 of Nb.BbvCI and 2 μL dNTPs. The experiment was performed at 37 °C for 1.5 h and the reaction was terminated by heating the mixture at 80 °C for 20 min to inactivate the enzymes. 2.5. Inhibition assay The inhibition of the drugs (gentamycin, benzylpenicillin, 5-fluorouracil, and mitomycin) on the activity of Dam MTase was investigated in the following experiment. First, 1 μM disparate drugs were separately mixed with the duplex substrate, 8 U/mL of Dam MTase, 1 μL of 10 Dam buffer, 160 μM SAM, after incubating the mixture for 2 h at 37 °C, 4 μL of 10 CutSmart buffer and 20 U Dpn I was added. The following procedures were the same as the analysis of Dam MTase. The effect of these inhibitors on the other enzymes including Dpn I, Klenow polymerase and NEase was shown in Supporting information. 2.6. Raman measurement The MBs incorporated with Raman probes were washed with PBS for three times to remove the additional reagents and redispersed in 15 μL of 0.01 M PBS (pH 7.4). Then 1.5 μL of each suspension was casted onto the gold chip and air-dried at room temperature before Raman analysis. The Raman spectra were measured by a Renisaw inVia Raman spectrometer at an excitation laser of 633 nm. The laser power was 5 mW, and the acquisition time for each spectrum was 5 s. Three repeated experiments were performed, the spectra were obtained from three different cites of each sample and calibrated with the WiRE Raman Software Version 3.3.
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2.7. Nondenaturing polyacrylamide gel electrophoresis (PAGE) The species produced in the process of DNA methylation and the strand-displacement amplification (SDA) were characterized by nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis (Acr¼acrylamide, Bis ¼N,N′-methylenebisacrylamide; Acr/Bis¼29/1). Tris–acetate-EDTA (TAE) (pH ¼8.5) was used as the separation buffer and the PAGE was generally run at 120 V for 1.5 h with loading of 10 μL of each sample into the lanes.
3. Results and discussion 3.1. The design principle of the SERS method for DNA MTase assay As models, DNA adenine methylation (Dam) MTase and Dpn I endonuclease are used in our experiments, both having the same special recognition sequence of the symmetric tetranucleotide 5′G-A-T-C-3′. Scheme 1 depicts the principle for analyzing the activity of Dam MTase. The duplex substrate is formed by the hybridization of DNA strands (S1 and S2). In the presence of Dam MTase, the symmetric 5′-G-A-T-C-3′of the substrate can be recognized and Dam MTase catalyzes the transfer of a methyl group from SAM to the N6 position of the adenine residues. Then, the methylated duplex substrate is cleaved into two parts by the methylation-sensitive restriction endonuclease Dpn I. In each part, there are only 6-complementary bases, so they are rapidly changed into two longer single-stranded DNAs (ssDNA, 12-base) and two shorter ssDNAs (6-base). Then the 12-base ssDNAs ingeniously functions as the primers (P1 and P2 in Scheme 1) of the strand-displacement amplification (SDA), which is achieved with
Scheme 1. Schematic Illustration of the proposed TPG-EXPAR strategy for Dam MTase activity assay.
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two repeated cycles of polymerization, nicking and displacement reactions, each is assisted by one primer. Once the primer (P1 or P2) hybridizes with the hairpin template (H1), it can extend along the template with the assistant of Klenow fragment polymerase and form a double-stranded DNA (dsDNA) containing the recognition site of nicking endonuclease (NEase). Subsequent singlestranded nicking produces a new replication site for polymerase and new ssDNAs (P3) in large quantities are released. Then P3 hybridizes with the loop part of hairpin template (H2) immobilized on the surface of magnetic beads (MBs–COOH). With the extension of P3, the hairpin structure of H2 is opened and the capture DNA (S3) conjugated on the Raman probe is attached to the opened stem and functions as a primer to trigger a new strand-displacement amplification. In the process of primer extension, a large number of Raman probes can be anchored on the surface of MBs by forming double-stranded DNAs and used for SERS measurement. 3.2. Feasibility of the assay The feasibility of our method for the assay of Dam MTase activity was evaluated by conducting a series of control experiments and the detection was based on the SERS signals of the Rox reporter on the Raman probes. In the absence of either Dam MTase or Dpn I, the duplex substrate could not be cleaved and no primer was produced for cycle amplification reaction, so the Raman signals were hardly observed (Fig. 1, curve a and b). Similarly, in the absence of either polymerase or NEase, SDA could not be performed and no Raman probes could be anchored on the surface of MBs, so the Raman signals were also very low only due to the nonspecific adsorption (Fig. 1, curve c and d). In contrast, in the presence of Dam MTase, Dpn I, polymerase and NEase, the Raman intensity was greatly raised (Fig. 1, curve e), which confirmed the feasibility of the proposed method for the assay of Dam MTase activity. For further confirming the feasibility of the method, we performed the experiment of nondenaturing polyacrylamide gel electrophoresis (PAGE) analysis. The variation of DNA in the processes of DNA methylation and the strand-displacement amplification (SDA) was shown in Fig. 2. Compared to lane 1 (the duplex of S1 and S2), there was a new band appeared in lane 2 when both Dam MTase and Dpn I were added to the test solution of S1 and S2, indicating that a methylation reaction has taken place and the methylated DNA was then cut into small pieces. The product (P1
Fig. 1. SERS spectra obtained by a series of control experiments: in the presence of Dpn I, polymerase and NEase (a), Dam, polymerase and NEase (b), Dam, Dpn I and NEase (c), Dam, Dpn I and polymerase (d), Dam, Dpn I, polymerase and NEase (e).
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Fig. 2. Polyacrylamide gel electrophoresis (PAGE) of the DNA ladder marker (M); the duplex substrate (S1 and S2) (1); the duplex substrate treated with Dam and Dpn I (2); the SDA product of the hairpin probe (H1) and P1, P2 (the product of methylation reaction); the hairpin probe, H1 (4).
and P2 in Scheme 1) of methylation reaction was then used to demonstrate the validity of strand-displacement amplification (SDA). In lane 4, there was an obvious migration band corresponded to the hairpin template (H1). After mixed with P1, P2 (the product of methylation reaction), NEase, polymerase and dNTPs, following by incubating the mixture at 37 °C for 90 min, the product was used for PAGE and the result was shown in lane 3 (Fig. 2). It could be seen that two new migration bands appeared, which indicated that SDA was performed very well. 3.3. Analytical performance For investigating the analytical performance of this strategy, different concentrations of Dam MTase were determined under the optimal experimental conditions. Fig. 3A showed the variance of Raman scattering intensity with the concentration of Dam MTase. It could be seen that the representative vibrational peaks of Rox (1499 cm 1 and 1644 cm 1) were displayed and the corresponding Raman intensity at 1499 cm 1 was utilized to quantitatively evaluate the Raman response to Dam MTase activity. As could be seen from Fig. 3B, the Raman intensity had a good linear fit to the logarithm of Dam MTase over a range of 4 orders of magnitude from 0.001 to 10 U/mL. The regression equation is ΔI ¼0.240 lgC þ0.762 (ΔI is the normalized Raman intensity subtracting the blank and C is the concentration of Dam MTase), and the correlation coefficient (R) is 0.992. A detection limit of 2.57 10 4 U mL 1 can be estimated using a S/N (signal/noise)
Fig. 3. (A) Raman spectra in response to different amounts of Dam MTase: 0, 0.001, 0.005, 0.01, 0.05, 0.1, 1, 10 U mL 1 from curve a to h; (B) The calibration curve of relative Raman intensity versus the amount of Dam MTase. Error bars showed the standard deviation of three experiments.
ratio of 3. A series of eleven repetitive measurements of 0.01 U/mL Dam MTase were used for estimating the precision, and the relative standard deviation (RSD) was 5.9%, showing good reproducibility. The sensitivity of the proposed strategy has dramatically improved as compared with most of the other developed approaches and the comparison of different methods for Dam MTase activity detection is shown in Table S2 (Supporting information). The detection limit is 3 orders of magnitude lower than that of the hybridization chain reaction-based branched rolling circle amplification method (Bi et al., 2013), 2 orders of magnitude lower than that of the exonuclease-mediated target recycling based fluorescence detection (Xing et al., 2014) and comparable with that of hairpin probe-based primer generation rolling circle amplification method (Zeng et al., 2013). The high sensitivity could be attributed to the following factors. First, three strand-displacement amplification modes are existed in this method, making a large number of Raman probes be anchored on the surface of MBs for SERS detection. Second, AuNPs are used for preparing the Raman probes, which can not only load more Rox-DNA but also enhance the SERS signal. Third, magnetic separation can easily circumvent the high background by removing the excess regents.
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Fig. 4. Selectivity of the proposed method. The concentration of Dam MTase is 10 U mL 1, and the concentration of M.SssI and HhaI MTase is 50 U mL 1.
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Fig. 5. (A) Influence of different drugs on the activity of Dam MTase. (1) No drug, (2) gentamycin, (3) 5-fluorouracil, (4) benzylpenicillin, (5) mitomycin.
3.4. Specificity of the method for dam MTase detection Furthermore, we investigated the specificity of this assay for Dam MTase. In this respect, M.Sss I and HhaI MTase were introduced as interference methyltranferases, which methylates all cytosine residues within the double-stranded dinucleotide recognition sequence 5′-C-G-3′ and the internal cytosine residue of the sequence 5′-G-C-G-C-3′, respectively. As shown in Fig. 4, a significantly higher Raman signal was observed in the presence of Dam MTase, while M.Sss I and HhaI have no obvious Raman enhancement compared with that of the blank test, even when adding 5-fold more of Dam MTase. These results demonstrated that the proposed strategy exhibited high selectivity due to the specific site recognition of Dam MTase toward the duplex substrate. 3.5. Inhibition assay Dam MTase plays an important role in the virulence of bacterial pathogens and DNA methylation is closely related to our health and disease, thus screening of inhibitors for DNA MTase receives more and more interest. In this research, for checking whether our sensitive method can be employed for high-throughput screen of Dam MTase inhibitors, some drugs (gentamycin, benzylpenicillin, 5-fluorouracil, and mitomycin) were investigated. From Fig. 5, it could be seen that gentamycin, 5-fluorouracil and benzylpenicillin could distinctly inhibit the Dam MTase activity and gentamycin was the most serious one with the inhibition ratio of about 45%. However, mitomycin had almost no effect on the methylation. These results indicate that our method has the potential to screen the drugs as inhibitors of Dam MTase. On the other hand, besides Dam, Dpn I, Klenow polymerase and NEase (Nb.BbvCI) are involved in our assay system, so it is necessary to examine the effect of these inhibitors on the other enzymes. The control experiments and the corresponding results were shown in Supporting information (Figs. S7 and S8), which indicated that no obvious influence could be observed when the concentration of the drugs was 1 mM.
were spiked into PBS and normal human serum (20%) respectively. The recoveries obtained by comparing the measured amounts to that of added Dam MTase were found to vary from 93.4% to 100.8%. (see Supporting information, Table S3) Three replicate determinations at different concentration levels exhibited RSDs ranging from 5.2% to 6.9% (n ¼3). The results showed an acceptable coherence between the data obtained in diluted serum and those in PBS, confirming the potentiality of this sensing system for MTase detection in real samples with other potentially competing species coexisting.
4. Conclusions In summary, a novel SERS method of DNA MTase assay has been developed by using of a target triggering primer generationmultiple signal amplification reaction. Taking advantages of the specific recognition of Dam MTase and DpnI, high amplification efficiency of the strategy and easy operation of SERS detection, a wide dynamic range and remarkably high sensitivity are obtained. Compared with previously reported methods, the detection limit of 2.57 10 4 U mL 1 is very low and other methyltranferases can be easily discriminated from Dam MTase with high selectivity. In addition, this sensitive assay method is successfully applied in the screening of inhibitors for Dam MTase. Given the attractive analytical characteristics, this sensing strategy might hold great promise for DNA methylation related clinical diagnosis and other biomedical research.
Acknowledgment This work was financially supported by the National Natural Science Foundation of China (21305072, 21275003).
3.6. Determination of dam MTase in real sample Appendix A. Supplementary material For testing the availability of this proposed method in real samples, the performance of Dam MTase in a human real serum sample was implemented. Different concentrations of Dam MTase
Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.02.057.
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