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Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity Gang Lin Wang, Hong Qun Luo and Nian Bing Li*
Received 28th January 2014 Accepted 19th June 2014
A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit
DOI: 10.1039/c4an00206g www.rsc.org/analyst
of 0.25 U mL1, which might make this method a good candidate for monitoring DNA methylation in the future.
1. Introduction DNA methylation, a well-known epigenetic event, plays an essential role in regulating gene transcription, cell growth and proliferation.1 It has been well-conrmed that DNA methylation is carried out with the aid of methyltransferase (MTase) using Sadenosyl-L-methionine (SAMe) as the methyl donor.2 Aberrant gene-specic methylation can signicantly alter gene expression and lead to numerous diseases.3 Thus, the development of simple, sensitive, and effective methods for DNA methylation detection and DNA MTase activity assay would be valuable for the diagnosis of genetic disease. Currently, several new methods have been reported for the detection of DNA methylation, including polymerase chain reaction-based techniques,4,5 high-performance liquid chromatography,6 capillary electrophoresis,7 gel electrophoresis,8 electrochemistry,9–14 DNA microarray,15 light scattering,16 colorimetry,17 and uorescence methods.18–20 Interestingly, with the development of materials science and nanotechnology, several nanomaterials, such as gold nanoparticles,21,22 magnetic nanoparticles,23 graphene,24 and carbon nanoparticles,25 are exploited for the quantication of methylation. Most of these methods, however, have the limitation of being time-intensive, DNA-consuming, requiring laborious treatment, and usually having a radiolabeling substrate requirement. Therefore, it is highly necessary to develop sensitive, simple, and effective methods for the detection of DNA methylation and the assay of MTase activity. Recently, gold nanorods (AuNRs) received widespread interest because of their anisotropic conguration and unique optical properties. The AuNRs have been applied for chemical
Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. E-mail: [email protected]; Fax: +86-23-6825-3237; Tel: +86-23-6825-3237
4572 | Analyst, 2014, 139, 4572–4577
and biochemical sensing with applications, including sensing metal ions,26,27 antibodies,28,29 DNA,30,31 and in photo-thermal therapy.32,33 More recently, the interaction between DNA and AuNRs has attracted signicant attention and has undergone substantial growth due to their collective behaviour. Herein, we proposed a simple, sensitive and cost-effective homogeneous assay for methyltransferase activity and inhibition study that utilizes uorescence resonance energy transfer (FRET) between AuNRs and uorescein (FAM)-tagged singlestranded DNA. As illustrated in Scheme 1, the assay is based on the fact that positively charged AuNRs have a higher affinity for double-stranded DNA (dsDNA) than for single-stranded DNA (ssDNA) because the surface charge density of dsDNA is signicantly larger than that of ssDNA. Dam MTase and Dpn I were chosen as the model MTase and restriction endonuclease, and a FAM-labelled DNA (FDNA1) and complementary DNA (cDNA1) were chosen as the substrate DNA. In the absence of methyltransferase, the uorescence of FDNA1 was quenched
A schematic illustration of the ultrasensitive detection of DNA methylation by gold-nanorods-based fluorescence resonance energy transfer (FRET) assay. Scheme 1
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because of the electrostatic attraction-based FRET between AuNRs and FDNA1. The formation of FDNA1/cDNA1 hybrid enhanced the electrostatic interaction between AuNRs and DNA, leading to an increase in FRET efficiency and a signicant decrease in uorescence intensity. However, when FDNA1/ cDNA1 was treated with Dam MTase, methylate was generated. Fluorescence enhancement was observed on further treatment with Dpn I, which cleaved FDNA1/cDNA1 hybrid substrate into small fragments. Because the electrostatic interaction between AuNRs and small DNA fragments is weaker than that between FDNA1 and cDNA1, the uorescence of the system was restored.
2. 2.1
Experimental section Reagents and materials
Tetrachloroauric acid trihydrate (HAuCl4$3H2O), sodium borohydride (NaBH4), cetyltrimethylammonium bromide (CTAB), and ascorbic acid were purchased from Chengdu Chemical Reagent Co., Chengdu, China. All the chemicals were of analytical reagent grade and were used without further purication. The DNA adenine methylation methyltransferase (Escherichia coli), Dpn I endonuclease, S-adenosyl-L-methionine (SAMe), and the corresponding buffer solutions were purchased from New England Biolabs (NEB, UK). All these standard solutions were prepared under 4 C and stored at 20 C. DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China. DNA stock solutions (1 mM) were prepared using 10 mM Tris–HCl buffer solution (pH 7.4) containing 0.5 M NaCl and maintained at 20 C. The sequences of oligonucleotides used in this work are listed as follows: FAM-DNA1 (FDNA1): 50 -CCTTTTGATCATTTT-FAM-30 cDNA1: 30 -GGAAAACTAGTAAAA-50 cDNA2: 3GGAAAACTCGTAAAA-50 . 2.2
Apparatus
Fluorescence spectra were recorded using a Hitachi F-2700 uorescence spectrophotometer (Hitachi Ltd., Japan) equipped with a xenon lamp excitation source. The samples were excited at 480 nm with an emission range from 510 to 620 nm. pH values were measured using a pH-3C precision pH meter (Leici Device Factory of Shanghai, China). Ultraviolet-visible absorption spectra were measured using a Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., Ltd., China). Transmission electron microscopy (TEM) image was obtained with a JEM-2000 instrument at 200 kV. 2.3
room temperature for 2 h and then used for the subsequent growth of AuNRs. In the growth solution, CTAB (40 mL, 0.1 M) was mixed with HAuCl4 (1.7 mL, 10 mM) and AgNO3 (250 mL, 10 mM). Aer gentle mixing, 270 mL of ascorbic acid was slowly added to the solution. The solution immediately became colorless immediately aer the addition of the ascorbic acid. Finally, 0.42 mL of gold seed solution was added to the growth solution mentioned above to initiate the growth of AuNRs. The solution immediately turned blue purple immediately aer adding the gold seed solution, indicating the generation of AuNRs. The AuNRs solution was kept undisturbed at 30 C for at least 16 h. Aerwards, excess CTAB was removed from the solution by repetitive centrifugations at a rate of 10 000 rpm for 10 min, and AuNRs were re-dispersed in distilled water. The AuNRs were stored in a refrigerator at 4 C before use. The resulting AuNRs were characterized by transmission electron microscopy (TEM) and ultraviolet visible absorption spectroscopy (UV). The TEM image shows that the obtained AuNRs were nearly mono-dispersed with an average length of 30.24 nm and width of 9.73 nm (Fig. 1A). The UV-Vis absorption spectrum of the initial AuNRs exhibits two peaks at 513 nm and 763 nm (Fig. 1B). The concentration of AuNRs (0.36 nM) was estimated by UV/vis spectroscopy based on an extinction coefficient of 4.2 109 M1 cm1 at l ¼ 763 nm for AuNRs.35
2.4
Assay of Dam methyltransferase activity
A series of standard Dam MTase solutions were prepared from 0.5 to 200 U mL1. To protect the activity of Dam MTase, all the standard solutions were prepared at 4 C and then stored at 20 C. The methylation experiment was conducted at 37 C for 2 h, and incubated in 20 mL of Dam MTase buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, pH 7.5) containing 1 mM FDNA1, 1 mM cDNA2, 80 mM SAMe, 4 U of Dpn I endonuclease and different amounts of Dam MTase. When the dsDNA molecules were methylated by DNA MTase and cleaved by Dpn I, 10 mL of 0.36 nM as-prepared CTAB coated AuNRs suspension was added to the system. The mixture was sequentially added to 470 mL of 10 mM Tris–HCl buffer solution (pH 7.4) and incubated for 5 min. Fluorescence measurements were performed using a Hitachi F-2700 uorescence spectrophotometer at excitation and emission wavelengths of 480 and 528 nm, respectively.
Preparation of gold nanorods
AuNRs were synthesized by a chemical reduction process using a seeding growth method, as previously reported by Murphy.34 Briey, the seed solution was prepared by mixing HAuCl4 (250 mL, 10 mM) and CTAB (7.5 mL, 0.1 mM) in a beaker. Then, freshly prepared ice cold NaBH4 (600 mL, 10 mM) solution was added all at once under stirring for 2 min. The solution immediately turned brown aer adding NaBH4, indicating the formation of gold seeds. The seed solution was maintained at
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Fig. 1 (A) TEM image of AuNRs. Inset shows that the AuNRs have a length of 30.24 nm and a width of 9.73 nm. (B) The corresponding absorption spectrum of AuNRs.
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2.5
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Evaluation of DNA methylation inhibitor
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To further extend the potential application of this assay in the inhibition assay, the inuence of drugs on Dam MTase activity was investigated by using 5-uorouracil (anti-cancer drug) as a model inhibitor of Dam MTase. The inhibition experiments were similar to that described above, except that the drug was added along with Dam MTase.
3. 3.1
Result and discussion Sensing strategy
To the best of our knowledge, FRET will take place occurs when there is an appreciable overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. As illustrated in Fig. 2, the absorption spectrum of as-prepared AuNRs shows two absorption peaks at 513 and 763 nm, respectively. At the same time, the FAM-DNA possesses an emission maximum at 528 nm. Thus, AuNRs can act as electron acceptors to quench uorophores in the photoinduced electron transfer process because the AuNRs have an absorption peak, which overlaps with the emission peak of FAM. Fig. 3 shows the uorescence spectra under different conditions. If the solution only contained FDNA1, a strong uorescence signal was detected (curve a). In the absence of methyltransferase, the uorescence of FDNA1 was quenched because of electrostatic attraction-based FRET between AuNRs and FDNA1 (curve b). Upon the addition of complementary cDNA1 to FDNA1, the formation of FDNA1/cDNA1 hybrid enhanced the electrostatic interaction between AuNRs and the DNA, leading to an increase in FRET efficiency and a signicant decrease in uorescence intensity (curve c). However, if FDNA1/ cDNA1 was treated with Dam MTase, DNA hybrid was methylated by methyltransferase. Fluorescence enhancement was observed when the DNA hybrid was further treated with Dpn I, which cleaved FDNA1/cDNA1 hybrid substrate into small fragments, leading to the weakening of electrostatic interaction between AuNRs and small DNA fragments. Thus, the uorescence of the system was restored (curve d). By monitoring the change in uorescence, the activity of Dam MTase and its inhibition were systematically investigated, thus providing a
Fig. 3 Fluorescence spectra of (a) 40 nM FDNA1, (b) 40 nM FDNA1 + 10 mL of AuNRs, (c) 40 nM FDNA1 + 40 nM cDNA1 + 10 mL of AuNRs, (d) 40 nM FDNA1 + 40 nM cDNA1 + 50 U mL1 Dam MTase + 4 U Dpn I + 10 mL of AuNRs, and (e) 40 nM FDNA1 + 40 nM cDNA2 + 50 U mL1 Dam MTase + 4 U Dpn I + 10 mL of AuNRs. The methylation experiment was conducted at 37 C for 2 h.
practical tool for studying DNA methylation and the assay of activity of methyltransferase and its inhibition. The sensing mechanism has been was further veried by TEM images and uorescence anisotropy. It can be seen from images a and b in Fig. 4 that AuNRs are well-dispersed in the aqueous medium in the absence and presence of singlestranded DNA1, illustrating that the electrostatic interaction between single-stranded DNA1 and AuNRs is not strong enough to change the suspension state of AuNRs. However, AuNRs aggregated when DNA1/cDNA1 duplex formed in the presence of cDNA1 (see image c of Fig. 4). It was found that CTAB-coated AuNRs without any modication were stable for 4 months at 4 C. Therefore, the aggregation of AuNRs is largely because of the strengthened electrostatic interaction between dsDNA and AuNRs because dsDNA has a higher density of surface charge than ssDNA. However, the aggregation of AuNRs is signicantly reduced in the presence of Dam MTase and Dpn I endonuclease that can cleave the DNA1/cDNA1 hybrid at a specic recognition site, which is mainly attributed to the weakened electrostatic attraction between the short DNA fragment and AuNRs. This is in accordance with the uorescence measurements. Therefore,
TEM images of AuNRs (a), FDNA1/AuNRs (b), FDNA1/AuNRs/ cDNA1 (c) and AuNRs/DNA1/cDNA1 after incubation with 50 U mL1 Dam MTase and 4 U Dpn I (d). The concentration of AuNRs and FDNA1 is 0.0072 and 20 nM, respectively. The methylation experiment was conducted at 37 C for 2 h. Fig. 4
Absorption spectrum of AuNRs and emission spectrum of FDNA1. The concentration of AuNRs and FDNA1 is 0.36 and 20 nM, respectively.
Fig. 2
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this FRET system was again demonstrated to be reasonable and reliable for assaying the activity of DNA MTase.
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3.2
Optimization of experimental conditions
To the best of our knowledge, DNA methylation was affected by several molecular species. To achieve the best conditions for the performance of DNA methylation assay, several variables of the monitoring system should be optimized. First, the inuence of the amount of AuNRs on DNA methylation assay was investigated by comparing the FRET efficiency. The quenching rate (Q) and recovery rate (R) were calculated using the following equations: Q¼
F0 F Fa F 100% 100% R ¼ F0 F0 F
hinders the process of methylation.36 Fig. 5C shows the inuence of Mg2+ concentration on DNA methylation. The velocity of DNA methylation and cleavage reached the maximum when the Mg2+ concentration was 10 mM. Thus, the optimum concentration of Mg2+ for the system was 10 mM. Another important factor for DNA methylation is the concentration of SAMe. As can be seen in Fig. 5D, the methylation/cleavage velocities increased on varying the concentration of SAMe from 0 to 160 mM. The initial methylation/cleavage velocities increased slowly when the concentration of SAMe exceeded 80 mM. In addition, SAMe was not very stable during in vitro experiments. Therefore, the concentration of SAMe was optimized to 80 mM.
3.3
DNA adenine methylation methyltransferase detection
where F0 and F are the uorescence of FDNA/cDNA1 in the absence and presence of AuNRs, respectively. Fa is the uorescence of the FDNA/cDNA1 duplex and AuNRs system aer the addition of MTase and Dpn I. As shown in Fig. 5A, when 10 mL of 0.36 nM AuNRs was added to the measuring system, both the uorescence quenching and recovering efficiency attained a good response. Thus, the optimal amount of AuNRs is 10 mL. Second, the methylation time was studied. As illustrated in Fig. 5B, the uorescence intensities reached a maximum value at 120 min and almost remained constant aer that. Therefore, 120 min was chosen as the methylation time in the owing studies. It is reported that Mg2+ is an essential coenzyme for restriction endonuclease (including Dpn I endonuclease) and
Under optimized conditions, the uorescence spectra of the FDNA1/DNA1 probe at different concentrations of Dam MTase solutions were recorded, and are shown in Fig. 6A. It can be seen from Fig. 6A and B that uorescence intensity gradually increased with the increasing concentration of Dam MTase. This is consistent with the fact that in the presence of higher concentrations of Dam MTase, more dsDNA probes were methylated and cleaved into small ssDNA fragments by Dpn I, resulting in a decrease in electrostatic interaction with AuNRs, which led to the recovery of uorescence. As illustrated in Fig. 6B, the calibration curve (Fig. 6B, inset) shows that the uorescence intensity is proportional to the logarithmic value of Dam MTase concentration ranging from 0.5 to 20 U mL1
Effect of species on methylation. (A) Effect of the amount of AuNRs, (B) effect of methylation time, (C) effect of the concentration of Mg2+, and (D) effect of the concentration of SAMe. The concentrations of FDNA1 and cDNA1 were 40 nM. The methylation experiment was conducted at 37 C for 2 h. Relative initial velocity ¼ Fn/F00 , where Fn is the fluorescence of the methylation/cleavage reaction with different concentrations of Mg2+ and SAMe, and F00 is the fluorescence of methylation/cleavage reaction without Mg2+ and SAMe.
(A) Fluorescence emission spectra of the FDNA1/DNA1 probe with increasing concentration of Dam MTase. The concentrations of Dam (from bottom to top): 0, 0.5, 1.0, 2.5, 5, 10, 20, 30, 50, 75, 100, 150, 200 U mL1. (B) The response of fluorescence intensity versus the concentration of Dam MTase.
Fig. 5
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Fig. 6
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Acknowledgements
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This work was supported by the National Natural Science Foundation of China (nos 21273174 and 20975083) and the Municipal Science Foundation of Chongqing City (no. CSTC2013jjB00002).
Notes and references Fig. 7 Inhibition assay of the different concentrations of 5-fluorouracil on the activity of Dam MTase.
with a detection limit of 0.25 U mL1 at a signal/noise ratio of 3, which is low compared to some previous reports, suggesting that this method is efficient to detect DNA methylation and the assay activity of Dam MTase. 3.4
Investigation of the selectivity of the proposed method
To investigate the selectivity of the proposed method, a onebase mismatched single strand DNA (cDNA2) was chosen to evaluate the cleavage selectivity of Dpn I. As shown in Fig. 3 (curve e), unchanged uorescence intensity was observed in the FDNA1/cDNA2 system, indicating that Dpn I could not cleave FDNA1/cDNA2 hybrid because Dpn I can only recognize the sequence 50 -G-A-T-C-30 . This result demonstrates that the present approach has high sequence selectivity for Dam MTase. 3.5 Estimation of the inhibition of DNA adenine methylation methyltransferase activity In addition, we investigated whether our method can be applied for evaluating and screening Dam MTase inhibitors such as antibiotics and anticancer drugs. In the present work, 5-uorouracil, an anticancer drug, was chosen as a model inhibitor. As shown in Fig. 7, the relative activity of Dam MTase decreased with the increasing concentration of 5-uorouracil, indicating that the activity of Dam MTase was inhibited. This was in accordance with the dose-dependent inhibition of drugs. Therefore, the proposed method was available to screen the inhibitors to the DNA MTase.
4. Conclusions In summary, a uorescence method for the detection of DNA methylation and assay of methyltransferase activity is proposed with AuNRs as a uorescence quencher on the basis of FRET. The detection limit of the method is as low as 0.25 U mL1, which might promise this method to be a good candidate for monitoring DNA methylation in the future. Furthermore, the protocol may offer new prospects for evaluating and screening the inhibitors of methyltransferase, which might be a great help for the discovery of anticancer drugs.
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Gold nanorods-based FRET assay for ultrasensitive detection of DNA methylation and DNA methyltransferase activity Gang Lin Wang, Hong Qun Luo and Nian Bing Li*
Received 28th January 2014 Accepted 19th June 2014
A fluorescence method for the detection of DNA methylation and the assay of methyltransferase activity is proposed using gold nanorods as a fluorescence quencher on the basis of fluorescence resonance energy transfer. It is demonstrated that this method is capable of detecting methyltransferase with a detection limit
DOI: 10.1039/c4an00206g www.rsc.org/analyst
of 0.25 U mL1, which might make this method a good candidate for monitoring DNA methylation in the future.
1. Introduction DNA methylation, a well-known epigenetic event, plays an essential role in regulating gene transcription, cell growth and proliferation.1 It has been well-conrmed that DNA methylation is carried out with the aid of methyltransferase (MTase) using Sadenosyl-L-methionine (SAMe) as the methyl donor.2 Aberrant gene-specic methylation can signicantly alter gene expression and lead to numerous diseases.3 Thus, the development of simple, sensitive, and effective methods for DNA methylation detection and DNA MTase activity assay would be valuable for the diagnosis of genetic disease. Currently, several new methods have been reported for the detection of DNA methylation, including polymerase chain reaction-based techniques,4,5 high-performance liquid chromatography,6 capillary electrophoresis,7 gel electrophoresis,8 electrochemistry,9–14 DNA microarray,15 light scattering,16 colorimetry,17 and uorescence methods.18–20 Interestingly, with the development of materials science and nanotechnology, several nanomaterials, such as gold nanoparticles,21,22 magnetic nanoparticles,23 graphene,24 and carbon nanoparticles,25 are exploited for the quantication of methylation. Most of these methods, however, have the limitation of being time-intensive, DNA-consuming, requiring laborious treatment, and usually having a radiolabeling substrate requirement. Therefore, it is highly necessary to develop sensitive, simple, and effective methods for the detection of DNA methylation and the assay of MTase activity. Recently, gold nanorods (AuNRs) received widespread interest because of their anisotropic conguration and unique optical properties. The AuNRs have been applied for chemical
Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. E-mail: [email protected]; Fax: +86-23-6825-3237; Tel: +86-23-6825-3237
4572 | Analyst, 2014, 139, 4572–4577
and biochemical sensing with applications, including sensing metal ions,26,27 antibodies,28,29 DNA,30,31 and in photo-thermal therapy.32,33 More recently, the interaction between DNA and AuNRs has attracted signicant attention and has undergone substantial growth due to their collective behaviour. Herein, we proposed a simple, sensitive and cost-effective homogeneous assay for methyltransferase activity and inhibition study that utilizes uorescence resonance energy transfer (FRET) between AuNRs and uorescein (FAM)-tagged singlestranded DNA. As illustrated in Scheme 1, the assay is based on the fact that positively charged AuNRs have a higher affinity for double-stranded DNA (dsDNA) than for single-stranded DNA (ssDNA) because the surface charge density of dsDNA is signicantly larger than that of ssDNA. Dam MTase and Dpn I were chosen as the model MTase and restriction endonuclease, and a FAM-labelled DNA (FDNA1) and complementary DNA (cDNA1) were chosen as the substrate DNA. In the absence of methyltransferase, the uorescence of FDNA1 was quenched
A schematic illustration of the ultrasensitive detection of DNA methylation by gold-nanorods-based fluorescence resonance energy transfer (FRET) assay. Scheme 1
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because of the electrostatic attraction-based FRET between AuNRs and FDNA1. The formation of FDNA1/cDNA1 hybrid enhanced the electrostatic interaction between AuNRs and DNA, leading to an increase in FRET efficiency and a signicant decrease in uorescence intensity. However, when FDNA1/ cDNA1 was treated with Dam MTase, methylate was generated. Fluorescence enhancement was observed on further treatment with Dpn I, which cleaved FDNA1/cDNA1 hybrid substrate into small fragments. Because the electrostatic interaction between AuNRs and small DNA fragments is weaker than that between FDNA1 and cDNA1, the uorescence of the system was restored.
2. 2.1
Experimental section Reagents and materials
Tetrachloroauric acid trihydrate (HAuCl4$3H2O), sodium borohydride (NaBH4), cetyltrimethylammonium bromide (CTAB), and ascorbic acid were purchased from Chengdu Chemical Reagent Co., Chengdu, China. All the chemicals were of analytical reagent grade and were used without further purication. The DNA adenine methylation methyltransferase (Escherichia coli), Dpn I endonuclease, S-adenosyl-L-methionine (SAMe), and the corresponding buffer solutions were purchased from New England Biolabs (NEB, UK). All these standard solutions were prepared under 4 C and stored at 20 C. DNA oligonucleotides were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China. DNA stock solutions (1 mM) were prepared using 10 mM Tris–HCl buffer solution (pH 7.4) containing 0.5 M NaCl and maintained at 20 C. The sequences of oligonucleotides used in this work are listed as follows: FAM-DNA1 (FDNA1): 50 -CCTTTTGATCATTTT-FAM-30 cDNA1: 30 -GGAAAACTAGTAAAA-50 cDNA2: 3GGAAAACTCGTAAAA-50 . 2.2
Apparatus
Fluorescence spectra were recorded using a Hitachi F-2700 uorescence spectrophotometer (Hitachi Ltd., Japan) equipped with a xenon lamp excitation source. The samples were excited at 480 nm with an emission range from 510 to 620 nm. pH values were measured using a pH-3C precision pH meter (Leici Device Factory of Shanghai, China). Ultraviolet-visible absorption spectra were measured using a Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., Ltd., China). Transmission electron microscopy (TEM) image was obtained with a JEM-2000 instrument at 200 kV. 2.3
room temperature for 2 h and then used for the subsequent growth of AuNRs. In the growth solution, CTAB (40 mL, 0.1 M) was mixed with HAuCl4 (1.7 mL, 10 mM) and AgNO3 (250 mL, 10 mM). Aer gentle mixing, 270 mL of ascorbic acid was slowly added to the solution. The solution immediately became colorless immediately aer the addition of the ascorbic acid. Finally, 0.42 mL of gold seed solution was added to the growth solution mentioned above to initiate the growth of AuNRs. The solution immediately turned blue purple immediately aer adding the gold seed solution, indicating the generation of AuNRs. The AuNRs solution was kept undisturbed at 30 C for at least 16 h. Aerwards, excess CTAB was removed from the solution by repetitive centrifugations at a rate of 10 000 rpm for 10 min, and AuNRs were re-dispersed in distilled water. The AuNRs were stored in a refrigerator at 4 C before use. The resulting AuNRs were characterized by transmission electron microscopy (TEM) and ultraviolet visible absorption spectroscopy (UV). The TEM image shows that the obtained AuNRs were nearly mono-dispersed with an average length of 30.24 nm and width of 9.73 nm (Fig. 1A). The UV-Vis absorption spectrum of the initial AuNRs exhibits two peaks at 513 nm and 763 nm (Fig. 1B). The concentration of AuNRs (0.36 nM) was estimated by UV/vis spectroscopy based on an extinction coefficient of 4.2 109 M1 cm1 at l ¼ 763 nm for AuNRs.35
2.4
Assay of Dam methyltransferase activity
A series of standard Dam MTase solutions were prepared from 0.5 to 200 U mL1. To protect the activity of Dam MTase, all the standard solutions were prepared at 4 C and then stored at 20 C. The methylation experiment was conducted at 37 C for 2 h, and incubated in 20 mL of Dam MTase buffer (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2, pH 7.5) containing 1 mM FDNA1, 1 mM cDNA2, 80 mM SAMe, 4 U of Dpn I endonuclease and different amounts of Dam MTase. When the dsDNA molecules were methylated by DNA MTase and cleaved by Dpn I, 10 mL of 0.36 nM as-prepared CTAB coated AuNRs suspension was added to the system. The mixture was sequentially added to 470 mL of 10 mM Tris–HCl buffer solution (pH 7.4) and incubated for 5 min. Fluorescence measurements were performed using a Hitachi F-2700 uorescence spectrophotometer at excitation and emission wavelengths of 480 and 528 nm, respectively.
Preparation of gold nanorods
AuNRs were synthesized by a chemical reduction process using a seeding growth method, as previously reported by Murphy.34 Briey, the seed solution was prepared by mixing HAuCl4 (250 mL, 10 mM) and CTAB (7.5 mL, 0.1 mM) in a beaker. Then, freshly prepared ice cold NaBH4 (600 mL, 10 mM) solution was added all at once under stirring for 2 min. The solution immediately turned brown aer adding NaBH4, indicating the formation of gold seeds. The seed solution was maintained at
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Fig. 1 (A) TEM image of AuNRs. Inset shows that the AuNRs have a length of 30.24 nm and a width of 9.73 nm. (B) The corresponding absorption spectrum of AuNRs.
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2.5
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Evaluation of DNA methylation inhibitor
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To further extend the potential application of this assay in the inhibition assay, the inuence of drugs on Dam MTase activity was investigated by using 5-uorouracil (anti-cancer drug) as a model inhibitor of Dam MTase. The inhibition experiments were similar to that described above, except that the drug was added along with Dam MTase.
3. 3.1
Result and discussion Sensing strategy
To the best of our knowledge, FRET will take place occurs when there is an appreciable overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor. As illustrated in Fig. 2, the absorption spectrum of as-prepared AuNRs shows two absorption peaks at 513 and 763 nm, respectively. At the same time, the FAM-DNA possesses an emission maximum at 528 nm. Thus, AuNRs can act as electron acceptors to quench uorophores in the photoinduced electron transfer process because the AuNRs have an absorption peak, which overlaps with the emission peak of FAM. Fig. 3 shows the uorescence spectra under different conditions. If the solution only contained FDNA1, a strong uorescence signal was detected (curve a). In the absence of methyltransferase, the uorescence of FDNA1 was quenched because of electrostatic attraction-based FRET between AuNRs and FDNA1 (curve b). Upon the addition of complementary cDNA1 to FDNA1, the formation of FDNA1/cDNA1 hybrid enhanced the electrostatic interaction between AuNRs and the DNA, leading to an increase in FRET efficiency and a signicant decrease in uorescence intensity (curve c). However, if FDNA1/ cDNA1 was treated with Dam MTase, DNA hybrid was methylated by methyltransferase. Fluorescence enhancement was observed when the DNA hybrid was further treated with Dpn I, which cleaved FDNA1/cDNA1 hybrid substrate into small fragments, leading to the weakening of electrostatic interaction between AuNRs and small DNA fragments. Thus, the uorescence of the system was restored (curve d). By monitoring the change in uorescence, the activity of Dam MTase and its inhibition were systematically investigated, thus providing a
Fig. 3 Fluorescence spectra of (a) 40 nM FDNA1, (b) 40 nM FDNA1 + 10 mL of AuNRs, (c) 40 nM FDNA1 + 40 nM cDNA1 + 10 mL of AuNRs, (d) 40 nM FDNA1 + 40 nM cDNA1 + 50 U mL1 Dam MTase + 4 U Dpn I + 10 mL of AuNRs, and (e) 40 nM FDNA1 + 40 nM cDNA2 + 50 U mL1 Dam MTase + 4 U Dpn I + 10 mL of AuNRs. The methylation experiment was conducted at 37 C for 2 h.
practical tool for studying DNA methylation and the assay of activity of methyltransferase and its inhibition. The sensing mechanism has been was further veried by TEM images and uorescence anisotropy. It can be seen from images a and b in Fig. 4 that AuNRs are well-dispersed in the aqueous medium in the absence and presence of singlestranded DNA1, illustrating that the electrostatic interaction between single-stranded DNA1 and AuNRs is not strong enough to change the suspension state of AuNRs. However, AuNRs aggregated when DNA1/cDNA1 duplex formed in the presence of cDNA1 (see image c of Fig. 4). It was found that CTAB-coated AuNRs without any modication were stable for 4 months at 4 C. Therefore, the aggregation of AuNRs is largely because of the strengthened electrostatic interaction between dsDNA and AuNRs because dsDNA has a higher density of surface charge than ssDNA. However, the aggregation of AuNRs is signicantly reduced in the presence of Dam MTase and Dpn I endonuclease that can cleave the DNA1/cDNA1 hybrid at a specic recognition site, which is mainly attributed to the weakened electrostatic attraction between the short DNA fragment and AuNRs. This is in accordance with the uorescence measurements. Therefore,
TEM images of AuNRs (a), FDNA1/AuNRs (b), FDNA1/AuNRs/ cDNA1 (c) and AuNRs/DNA1/cDNA1 after incubation with 50 U mL1 Dam MTase and 4 U Dpn I (d). The concentration of AuNRs and FDNA1 is 0.0072 and 20 nM, respectively. The methylation experiment was conducted at 37 C for 2 h. Fig. 4
Absorption spectrum of AuNRs and emission spectrum of FDNA1. The concentration of AuNRs and FDNA1 is 0.36 and 20 nM, respectively.
Fig. 2
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this FRET system was again demonstrated to be reasonable and reliable for assaying the activity of DNA MTase.
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3.2
Optimization of experimental conditions
To the best of our knowledge, DNA methylation was affected by several molecular species. To achieve the best conditions for the performance of DNA methylation assay, several variables of the monitoring system should be optimized. First, the inuence of the amount of AuNRs on DNA methylation assay was investigated by comparing the FRET efficiency. The quenching rate (Q) and recovery rate (R) were calculated using the following equations: Q¼
F0 F Fa F 100% 100% R ¼ F0 F0 F
hinders the process of methylation.36 Fig. 5C shows the inuence of Mg2+ concentration on DNA methylation. The velocity of DNA methylation and cleavage reached the maximum when the Mg2+ concentration was 10 mM. Thus, the optimum concentration of Mg2+ for the system was 10 mM. Another important factor for DNA methylation is the concentration of SAMe. As can be seen in Fig. 5D, the methylation/cleavage velocities increased on varying the concentration of SAMe from 0 to 160 mM. The initial methylation/cleavage velocities increased slowly when the concentration of SAMe exceeded 80 mM. In addition, SAMe was not very stable during in vitro experiments. Therefore, the concentration of SAMe was optimized to 80 mM.
3.3
DNA adenine methylation methyltransferase detection
where F0 and F are the uorescence of FDNA/cDNA1 in the absence and presence of AuNRs, respectively. Fa is the uorescence of the FDNA/cDNA1 duplex and AuNRs system aer the addition of MTase and Dpn I. As shown in Fig. 5A, when 10 mL of 0.36 nM AuNRs was added to the measuring system, both the uorescence quenching and recovering efficiency attained a good response. Thus, the optimal amount of AuNRs is 10 mL. Second, the methylation time was studied. As illustrated in Fig. 5B, the uorescence intensities reached a maximum value at 120 min and almost remained constant aer that. Therefore, 120 min was chosen as the methylation time in the owing studies. It is reported that Mg2+ is an essential coenzyme for restriction endonuclease (including Dpn I endonuclease) and
Under optimized conditions, the uorescence spectra of the FDNA1/DNA1 probe at different concentrations of Dam MTase solutions were recorded, and are shown in Fig. 6A. It can be seen from Fig. 6A and B that uorescence intensity gradually increased with the increasing concentration of Dam MTase. This is consistent with the fact that in the presence of higher concentrations of Dam MTase, more dsDNA probes were methylated and cleaved into small ssDNA fragments by Dpn I, resulting in a decrease in electrostatic interaction with AuNRs, which led to the recovery of uorescence. As illustrated in Fig. 6B, the calibration curve (Fig. 6B, inset) shows that the uorescence intensity is proportional to the logarithmic value of Dam MTase concentration ranging from 0.5 to 20 U mL1
Effect of species on methylation. (A) Effect of the amount of AuNRs, (B) effect of methylation time, (C) effect of the concentration of Mg2+, and (D) effect of the concentration of SAMe. The concentrations of FDNA1 and cDNA1 were 40 nM. The methylation experiment was conducted at 37 C for 2 h. Relative initial velocity ¼ Fn/F00 , where Fn is the fluorescence of the methylation/cleavage reaction with different concentrations of Mg2+ and SAMe, and F00 is the fluorescence of methylation/cleavage reaction without Mg2+ and SAMe.
(A) Fluorescence emission spectra of the FDNA1/DNA1 probe with increasing concentration of Dam MTase. The concentrations of Dam (from bottom to top): 0, 0.5, 1.0, 2.5, 5, 10, 20, 30, 50, 75, 100, 150, 200 U mL1. (B) The response of fluorescence intensity versus the concentration of Dam MTase.
Fig. 5
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Fig. 6
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Acknowledgements
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This work was supported by the National Natural Science Foundation of China (nos 21273174 and 20975083) and the Municipal Science Foundation of Chongqing City (no. CSTC2013jjB00002).
Notes and references Fig. 7 Inhibition assay of the different concentrations of 5-fluorouracil on the activity of Dam MTase.
with a detection limit of 0.25 U mL1 at a signal/noise ratio of 3, which is low compared to some previous reports, suggesting that this method is efficient to detect DNA methylation and the assay activity of Dam MTase. 3.4
Investigation of the selectivity of the proposed method
To investigate the selectivity of the proposed method, a onebase mismatched single strand DNA (cDNA2) was chosen to evaluate the cleavage selectivity of Dpn I. As shown in Fig. 3 (curve e), unchanged uorescence intensity was observed in the FDNA1/cDNA2 system, indicating that Dpn I could not cleave FDNA1/cDNA2 hybrid because Dpn I can only recognize the sequence 50 -G-A-T-C-30 . This result demonstrates that the present approach has high sequence selectivity for Dam MTase. 3.5 Estimation of the inhibition of DNA adenine methylation methyltransferase activity In addition, we investigated whether our method can be applied for evaluating and screening Dam MTase inhibitors such as antibiotics and anticancer drugs. In the present work, 5-uorouracil, an anticancer drug, was chosen as a model inhibitor. As shown in Fig. 7, the relative activity of Dam MTase decreased with the increasing concentration of 5-uorouracil, indicating that the activity of Dam MTase was inhibited. This was in accordance with the dose-dependent inhibition of drugs. Therefore, the proposed method was available to screen the inhibitors to the DNA MTase.
4. Conclusions In summary, a uorescence method for the detection of DNA methylation and assay of methyltransferase activity is proposed with AuNRs as a uorescence quencher on the basis of FRET. The detection limit of the method is as low as 0.25 U mL1, which might promise this method to be a good candidate for monitoring DNA methylation in the future. Furthermore, the protocol may offer new prospects for evaluating and screening the inhibitors of methyltransferase, which might be a great help for the discovery of anticancer drugs.
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