Analyst View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
PAPER
Cite this: DOI: 10.1039/c4an01359j
View Journal
Label-free fluorescence detection of DNA methylation and methyltransferase activity based on restriction endonuclease HpaII and exonuclease III Chunyan Gao, Henan Li, Yuanjian Liu, Wei Wei,* Yuanjian Zhang and Songqin Liu Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL1 with a detection limit of 0.16 U mL1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical
Received 24th July 2014 Accepted 24th September 2014
triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no
DOI: 10.1039/c4an01359j
obvious effects on the assay performance, indicating that the method has great potential for further
www.rsc.org/analyst
application in complex samples.
Introduction DNA methylation plays an important role in the regulation of gene transcription, eukaryote development, and cellular processes as well as the pathogenesis of various human diseases such as cancers.1,2 Especially, methylation of cytosine (C) in the specic DNA sequence is an important epigenetic modication,3,4 which has frequently occurred at the carbon 5 position of C in CpG islands (50 -CG-30 ).5–7 In normal cells, most CpG islands spanning the promoter regions are unmethylated, so their downstream genes are active. However, the CpG islands in cancer cells are methylated, and their downstream genes are silenced.8 Abnormal methylation in CpG islands can result in the change of normal cellular functions and phenotypes,9 and has been considered as a potential biomarker of diseases. Thus, a simple system is needed to recognize methylated specic DNA
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Chemistry and Chemical Engineering, Southeast University, Jiangning District, Nanjing, 211189, P.R. China. E-mail: [email protected]; Fax: +86-25-5209061; Tel: +86-25-52090613
This journal is © The Royal Society of Chemistry 2014
sequences and be applied to early diseases diagnostics and determine cancer types. The conventional sequencing technique cannot be applied to gene-specic methylation detection, owing to methylcytosine (mC) and C displaying identical Watson–Crick base-pair behavior.10 Currently, the methods for detection of DNA methylation are mainly based on distinguishing between mC and C in DNA, including methylation-sensitivity restriction enzyme digestion,11,12 bisulte treatment,13,14 methylation antibody recognition,15,16 and various polymerase chain reaction (PCR) techniques.17 The assay methods based on bisulte and restriction enzymes are widely used to distinguish mC and C in DNA. In the bisulte-based assay, C is deaminated and then converted to uracil (U) through the treatment of bisulte, whereas mC remains almost unchanged due to the extremely low reactivity. The bisulte-based method is currently considered a gold standard assay, yet suffers signicantly from complicated operations and high equipment cost.18 In the restriction enzyme assay, the restriction enzymes sensitively recognize the methylation position and selectively catalyze the scission of the specic DNA sequence. When the C in the specic sequence is methylated, the cleavage is inhibited.12
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
Recently, Wang and his co-workers reported that a methylationsensitive restriction enzyme was used to digest the genic DNA. Only methylated DNA can be amplied by PCR and the methylation level was detected through uorescence resonance energy transfer (FRET) between the cationic conjugated polymer (CCP) and uorescein labeled dNTP of DNA.19 This can provide an economical method to identify the specic site of methylation in DNA and survey the extent of DNA methylation. Moreover, analyzing the MTase activity is also important in the regions of clinical diagnostics because DNA MTase catalyzes the covalent addition of the methyl group to C in DNA in the formation of mC.20,21 Therefore, a fast, cheap, simple, and selective method for detection of DNA methylation and M.SssI MTase activity is imperative. Exonuclease III (Exo III) is a kind of exonuclease that has high exodeoxyribonuclease activity for dsDNA to catalyze the stepwise removal of mononucleotides from the 30 to 50 terminus and limited activity on 30 -overhang ends of dsDNA or ssDNA.22 More recently, Exo III has been extensively used to detect target DNA sensitively through signal amplication. For example, Xiao and co-workers have designed an amplied DNA detection method employing a stem-loop DNA molecular beacon as the signal probe through the cleavage function of exonuclease III on dsDNA.23 Here, the cleavage function of exonuclease III combined with endonuclease HpaII was applied to detect DNA methylation and M.SssI MTase activity. Based on the obvious different uorescence properties of TO in the presence of ssDNA or dsDNA, the method for detection of DNA methylation and M.SssI MTase activity is designed (Scheme 1). The probe and target DNA hybridize to form dsDNA with two 30 protruding termini, which are resistant to cleavage by Exo III. HpaII endonuclease is used to identify the 50 -CCGG-30 sequences and cleave them at the desired position (bottom in Scheme 1), then Exo III could identify the recessed 30 -hydroxyl termini of dsDNA and catalyze the stepwise removal of mononucleotides from this position. The uorescence intensity of the added TO is weak because only ssDNA is contained in the solution (Scheme 1a). If dsDNA was methylated by M.SssI MTase, the function of HpaII was
Paper
inhibited because the cleavage is blocked by CpG methylation, as a result, Exo III cannot work and the dsDNA maintains its integrity. The added TO intercalated into its double strand and showed strong uorescence intensity (Scheme 1b). The uorescence intensity of TO has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL1 with a detection limit of 0.16 U mL1. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These demonstrated that this sensor used for bio-monitoring of site specic DNA methylation and sensitive detection of M.SssI activity is simple, fast and cheap.
Experimental section Chemicals S-Adenosylmethionine (SAM), CpG methyltransferase (M.SssI MTase), restriction endonucleases HpaII and MspI (HpaII) were supplied by Thermo Scientic (USA). Exonuclease III (Exo III) was purchased from TAKARA biotechnology Co., Ltd. (Dalian, China) and used without further purication. Human serum was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Aladdin reagent Co., Ltd (Shanghai, China) and acetaldehyde (CH3CHO) was obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris), HCl, NaCl, hydrogen peroxide (30% in water) (H2O2), ascorbic acid, ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-2Na), and MgCl2$6H2O were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DL-dithiothreitol (DTT), bovine serum (BSA) and thiazole orange (TO) were of analytical grade and obtained from Sigma-Aldrich. The stock solution of 20 mM TO was prepared in dimethylsulfoxide and diluted with deionized water to a desired concentration. All oligonucleotides were obtained from Shanghai Sangon biotechnology Co., Ltd (Shanghai China). Then, the sequences include single-stranded 32 mer probe DNA (S1: 50 -TCC TGG GAG AGA CCG GCG CAC AGA GGT TAT TA-30 ), a fragment as part of p53 (S2: 50 -CCT CTG TGC GCC GGT CTC TCC CAG GAC AGG CA-30 , target DNA), one base-mismatched target DNA (S3: 50 -CCT CTG TGC GCT GGT CTC TCC CAG GAC AGG CA-30 ), and unrelated DNA (50 -AG ACA GAG GAA AGA AAT TCT CGA AAA ATT ATC-30 ). All solutions of the experiments were prepared with double distilled water. Apparatus All uorescence spectra were obtained using a Fluoromax 4 spectrouorometer (Horiba, Japan), and recorded upon 480 nm excitation with 0.1 s integration time and a 5 nm integration step at room temperature. Hybridization of S1 with target DNA
Scheme 1 Schematic illustration of the principle of label-free fluorescence detection of site-specific DNA methylation and M.SssI MTase activity based on HpaII and Exo III.
Analyst
100 nM probe DNA (S1) and 100 nM target DNA (S2) were incubated in 50 mM Tris–HCl buffer solution (pH 7.9) including 100 mM NaCl and 5 mM MgCl2 at 70 C for 10 min. Then, the solution was cooled down to room temperature. Aer a specic incubation period, TO was added to the solution. Next, the
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
solution was allowed to stand at room temperature for 1 hour, and the uorescence spectra were recorded.
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Methylation of CpG and cleavage of HpaII endonuclease and Exo III The methylation of the S1 (100 nM)/S2 (100 nM) hybrid was performed at 37 C for 1 h in 50 mM Tris–HCl buffer (pH 7.9) containing 50 mM SAM, 100 mM NaCl, 5 mM MgCl2, 0.1 mg mL1 BSA, 1 mM DTT and various concentrations of M.SssI MTase (from 0 to 16 U mL1). Aer methylation, 10 U HpaII and 25 U Exo III were added to the above solution, aerward, cleavage was carried out at 37 C for 2.5 h. Next, 2.5 mM TO was added, the mixture was allowed to stand for 1 h, and the uorescence spectra were recorded without further purication at room temperature. 90 nM unrelated DNA was added into the above S1/S2 hybrid to study their interference with the method. Methylation of cytosine by a hydroxyl radical triggered by DMSO and CH3CHO 100 nM S2 was rst treated with a chemical reagent (100 mM DMSO or CH3CHO in the presence of Fenton reagent (25.0 mM FeSO4, 98.0 mM H2O2, 53.5 mM ascorbic acid, and 15.0 mM EDTA2Na)) through vigorous shaking at room temperature for 3 h, and then it was separated by using Millipore's Amicon Ultra-0.5 centrifugal lter device to remove excessive chemical reagent. The reaction was performed under a N2 atmosphere and the solution is 50 mM Tris–HCl (pH 7.3) containing 100 mM NaCl. Aer that, S1 hybridized with treated S2 in 50 mM Tris–HCl (pH 7.9) containing 100 mM NaCl and 5 mM MgCl2, which was incubated at 70 C for 10 min, and then the solution was cooled down to room temperature. Then, 10 U HpaII and 25 U Exo III were added and cleavage was performed at 37 C for 2.5 h. Finally, 2.5 mM TO was added, the mixture was allowed to stand for 1 h and the uorescence spectra were recorded without further purication at room temperature. The total reaction volume was 500 mL.
Analyst
(S2) and its complementary sequences were designed as probe DNA (S1). TO in an aqueous solution emits almost no uorescence (Fig. 1, curve a). In the presence of probe DNA, TO is adsorbed onto ssDNA through electrostatic attraction, the uorescence of TO has a weak signal (Fig. 1, curve b). In the presence of target DNA, the hybrid of S1/S2 was formed, and TO intercalated into the minor groove of the hybrid by electrostatic interactions. The structure of TO tends to be planar due to the steric effect,25,26 resulting in obvious enhancement of the uorescence signal (Fig. 1, curve c). The uorescence signal is related to the TO concentration, and the optimization of TO concentration is shown in Fig. 2. Both of the uorescence intensities of TO–S1 (Fig. 2, curve a) and TO–S1/S2 (Fig. 2, curve b) increased with the increasing TO concentration. The change in uorescence signal (Fb Fa) also increased along with the increasing TO concentration, tending to balance at 2.5 mM. In order to obtain the highest sensitivity, 2.5 mM was chosen as the optimum TO concentration. The function of HpaII and Exo III and the optimization of their concentration Aer the addition of HpaII into the S1/S2 hybrid solution, the uorescence intensity of TO has no obvious change, indicating that the two parts of the S1/S2 hybrid cleaved by HpaII still retained their duplex form (Fig. 1, curve d). Upon the addition of
Detection M.SssI activity in human serum samples 500 nM S1/S2 hybrid was added to 100 mL of the human serum sample. Then, DNA was extracted from the above sample with a nucleic acid automatic extraction system (Applied Biosystems, Thermo Fisher Scientic Oy, USA) and was diluted 5 times with 50 mM Tris–HCl buffer before use. In addition, 100 nM S1/S2 hybrid added to a 10-fold diluted human serum sample without extraction was used as a control.
Fig. 1 Fluorescence spectra (left panel) and histogram (right panel) of 2.5 mM TO (a), TO in the presence of S1 (b), S1/S2 (c), S1/S2 and HpaII (d), S1/S2, HpaII and Exo III (e), S1/S2 and Exo III (f), S1/S2, M.SssI, HpaII and Exo III (g) and S1/S2, M.SssI, MspI and Exo III (h).
Results and discussion Fluorescence properties of TO and its concentration optimization p53 has proved to be a tumor-suppressor gene. The methylation of the region of p53 plays a key role in the development of at least 60% of tumors of the colon, breast, lung, ovaries, cervix, adrenal cortex, bone, and bladder and at least 30% of brain tumors.24 So, a part of sequences of the p53 gene containing a specic methylation site (50 -CCGG-30 ) was chosen as target DNA
This journal is © The Royal Society of Chemistry 2014
Fig. 2 Fluorescence intensity of various concentrations of TO in the presence of S1 (a), S1/S2 (b) and Fb Fa (c). Fa is the fluorescence intensity of TO–S1 and Fb is the fluorescence intensity of the TO–S1/S2.
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
both Exo III and HpaII into the S1/S2 hybrid solution, the uorescence intensity of TO decreased nearly to that in the ssDNA (S1), which proved the digestion function of Exo III (Fig. 1, curve e). When only Exo III was added to the S1/S2 hybrid solution (Fig. 1, curve f), the TO intensity did not change, indicating that the cleavage function of HpaII is efficient and necessary. The effects of HpaII and Exo III concentration and reaction time were investigated. The uorescence intensity of TO decreased with the increasing concentration of HpaII in the range of 0–20 U mL1. Further increase of HpaII concentration caused little enhancement of the uorescence signal in the range of 20–60 U mL1. Therefore, 20 U mL1 HpaII was chosen as the optimum concentration for all measurements (Fig. 3A). In the presence of 20 U mL1 HpaII, the uorescence intensity of TO decreased with the increasing concentration of Exo III and tended to a plateau at 50 U mL1, indicating that the maximum degradation efficiency was obtained. Therefore, 50 U mL1 was chosen in the following experiments (Fig. 3B). The reaction time of HpaII and Exo III also affected the uorescence signal, which reduced sufficiently with increasing reaction time and had a tendency to a constant value at 140 min (Fig. 3C). As a result, 150 min was chosen as the optimal reaction time. Detection of the target DNA and its selectivity The quantitative detection of DNA by the proposed uorescence sensor was investigated under the optimized conditions. As
Paper
shown in Fig. 4A, the uorescence intensity at 528 nm increased with the increasing concentration of target DNA from 0 nM to 140 nM. Fig. 4B depicts that the uorescence signal increased with the concentration of target DNA from 0 to 100 nM, and reached a plateau in the range of 100–140 nM. The linear correlation was obtained in the concentration range of 0.6 nM to 80 nM and the linear correlation was 0.992 (inset in Fig. 4B). The detection limit of 0.1 nM was obtained in terms of 3 times deviation of the blank sample. One base mismatched target DNA (S3) was used to evaluate the selectivity of the method. Compared with the uorescence intensity of TO–S1/S2 (Fig. 5a), the intensity of TO–S1/S3 (Fig. 5b) decreased only about 10%, indicating that TO has poor selectivity for the one base mismatched target DNA. When the S1/S2 hybrid was treated with 10 U HpaII and 25 U Exo III for 2.5 h at 37 C, the uorescence response of TO decreased by about 60% (Fig. 5c) compared with the uorescence intensity of TO–S1/S2. However, the intensity of TO in the presence of S1/S3 that was treated with 10 U HpaII and 25 U Exo III changed little compared with that not treated with HpaII and Exo III (Fig. 5d). This demonstrated that HpaII and Exo III do not work in the presence of one base mismatched target DNA, indicating that the proposed method based on TO (signal molecule), HpaII (cleavage of the ds DNA into two parts) and Exo III (degradation of dsDNA to ssDNA) has good selectivity to differentiate one base mismatched DNA from intact target DNA.
Detection of M. SssI activity
Fig. 3 The fluorescence intensity of TO as a function of the concentration of (A) HpaII, (B) Exo III, and (C) the reaction time of HpaII and Exo III.
Fig. 4 (A) Corresponding fluorescence spectra of TO in the presence of S1 and 0 nM, 0.6 nM, 4 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 120 nM and 140 nM target DNA (a–k), and (B) the standard curve for the relationship between the fluorescence signal and the concentration of target DNA, the inset shows a linear relationship between the fluorescence signal and the concentration of target DNA.
Analyst
It is known that in the presence of M.SssI MTase and a methyl group donor, S-adenosyl-L-methionine (SAM),27 the second cytosine at the sequence of 50 -CCGG-30 can be methylated to 50 -CmCGG-30 . The methylated cytosine prevents dsDNA from cutting by HpaII (ref. 28) and degrading by Exo III. In the presence of M.SssI MTase, the S1/S2 hybrid was methylated at the specic site of 50 -CG-30 , the treatment of HpaII and Exo III did not change the uorescence of the TO signal (Fig. 1, curve g). However, when the methylated S1/S2 was treated with restriction endonuclease MspI and Exo III, the uorescence signal of TO decreased (Fig. 1, curve h), because MspI is not sensitive to methylation, suggesting that isoschizomer MspI does not work in the experiments. Thus, the activity of M.SssI
Fig. 5 Fluorescence spectra (left panel) and histogram (right panel) of TO–S1/S2 (a), TO–S1/S3 (b), TO–S1/S2 (c) and TO–S1/S3 (d) incubated with HpaII and Exo III at 37 C for 2.5 h in 50 mM Tris–HCl (pH 7.9).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Paper
can be detected by comparing the uorescence of TO–S1/S2 in the presence of various concentrations of M.SssI. The methylation level was closely dependent on the reaction time of M.SssI, we rstly evaluate the effect of methylation time in the methylation process. The S1/S2 hybrid was methylated for various times under the function of M.SssI MTase, then the uorescence signal was recorded aer the cleavage of methylated hybrid by HpaII and Exo III (Fig. 6). The uorescence signal increased rapidly in the range of 0–40 min and tended to a plateau in the range of 40–120 min. Therefore, 1 h was chosen as the optimal time for full reaction. The S1/S2 hybrid was methylated with various concentrations of M.SssI MTase from 0 U mL1 to 16 U mL1 for 1 h, and then incubated with HpaII and Exo III. Aer reaction for 2.5 h, 2.5 mM TO was added to the solution. The intensity of uorescence signals increased with the increasing concentration of M.SssI MTase (Fig. 7A) because more S1/S2 hybrid is methylated and the cleavage and degradation functions of HpaII and Exo III are prevented at higher concentrations of M.SssI MTase. Fig. 7B reveals that the uorescence signals increased with the increasing concentration of M.SssI MTase from 0 to 12 U mL1, and reached a constant value at 12 U mL1. A linear correlation
Analyst
was obtained in the concentration range of 1 U mL1 to 10 U mL1 and the correlation coefficient was 0.997 (inset in Fig. 7B). The detection limit of 0.16 U mL1 was obtained in terms of 3 times deviation of the blank sample, which is lower than that of the chemiluminescence (CL) method (0.52 U mL1)29 and the label-free colorimetric method (0.4 U mL1).30 Thus, this proposed method can be applied to sensitive detection of MTase activity. Detection of cytosine methylation induced by a hydroxyl radical triggered by DMSO and CH3CHO The method has also been used to detect the methylation of DNA cytosine by DMSO and CH3CHO in the presence of Fenton reagent which provided a hydroxyl radical. The target DNA was treated with DMSO or CH3CHO in the presence Fenton reagent at room temperature for 3 h, then the mixture was separated through a centrifugal lter device (molecular weight 3 kD, centrifugal speed 14 000 rcf, 15 min). Aer ultraltration, S1 hybridized with treated S2, and then 10 U HpaII and 25 U Exo III were added for incubation. Fig. 8 shows that the uorescence intensity of HpaII and Exo III cut S1/S2 hybrid (Fig. 8, curve b) was much lower than that of the S1/S2 hybrid (Fig. 8, curve a). When S2 was treated with CH3CHO (Fig. 8A, curve c) or DMSO (Fig. 8A, curve d) in the presence of Fenton reagent, the uorescence intensity was stronger than that of intact S1/S2. These results indicated that these two chemical reagents methylated the target DNA, as a result, the functions of HpaII and Exo III are prevented. This was in agreement with the reported results that the methylation of cytosine occurred through methyl radicals produced by DMSO and CH3CHO in the presence of Fenton reagent.31 Therefore, this method can be used to detect the DNA methylation induced by chemical reagents. Application of the method in human serum samples
The fluorescence signal of TO was dependent on the methylation time with M.SssI. Fig. 6
Fig. 7 (A) The effect of M.SssI concentration on the fluorescence response of TO. The concentration of M.SssI is (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, (g) 6, (h) 8, (i) 9, (j) 10, (k) 12, and (l) 16 U mL1, respectively. Before the fluorescence spectra were recorded, the S1/S2 hybrid was methylated by M.SssI and 50 mM SAM for 1 h, and then incubated with HpaII and Exo III for 2.5 h, finally TO was added. (B) The plot of the fluorescence signal vs. M.SssI concentration, the inset: the linear relationship between the fluorescence signal and the concentration of the M.SssI concentration.
This journal is © The Royal Society of Chemistry 2014
The application of the method in complex biological matrices is another challenging factor to evaluate the performance of the sensor. 90 nM unrelated sequences were used as interference materials. As depicted in Fig. 9A, it was observed that 90 nM unrelated sequence has no obvious effect on the detection, however, the uorescence intensity for methylated DNA (c) and intact DNA (b) showed obvious differences. 10% diluted human
Fig. 8 Fluorescence spectra (left panel) and histogram (right panel) of TO–S1/S2 (a), TO–S1/S2 containing HpaII and Exo III (b), S2 was treated with Fenton reagent including CH3CHO (c), and Fenton reagent including DMSO (d) following the same procedures as that in (b).
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
Fig. 9 (A) Results obtained from (a) S1/S2, (b) S1/S2 with HpaII and Exo III, (c) S1/S2 with 7 U M.SssI MTase, HpaII and Exo III in the 50 mM Tris– HCl buffer solution (black column) and the 50 mM Tris–HCl buffer solution containing 90 nM unrelated DNA (red column). (B) The comparison of signals of (a) S1/S2, (b) S1/S2 with HpaII and Exo III, (c) S1/S2 with 7 U M.SssI MTase, HpaII and Exo III in 10% diluted human serum samples (red column), 20% diluted DNA extraction of human serum samples (blue column) and 50 mM Tris–HCl buffer solution (black column).
serum samples (red column) and 20% diluted DNA extraction samples (blue column) were investigated, respectively. Fig. 9B shows that the signal ratio of methylated DNA (b) to intact DNA (c) for black, red and blue columns were 2.5, 1.8 and 3, which indicated that the method can be used to differentiate the methylated DNA from intact DNA in human serum samples. It was also obvious that aer the DNA extraction, the assay of the human serum sample is more sensitive.
Conclusions To sum up, we have presented a label-free uorescence method to detect DNA methylation and M.SssI activity based on HpaII and Exo III. The method operates via directly detecting the TO uorescence intensity, provides a rapid, simple, homogeneous phase and site-specic methylation assay, and requires no bisulte change, PCR amplication, label probe and separation. The method also has good selectivity to differentiate one base mismatched target DNA from intact DNA. As the epigenetic basis of cancerous transformation remains to be discovered, the demand for effective and inexpensive strategies to detect human methyltransferase activity will continue to grow. Human serum samples have no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
Acknowledgements The project is supported by the National Natural Science Foundation of China (Grant no. 21175021, 21205014, and 21475020), and the Natural Science Foundation of Jiangsu province (BK2012734).
Notes and references 1 K. D. Robertson and A. P. Wolffe, Nat. Rev. Genet., 2000, 1, 11–19.
Analyst
Paper
2 M. Toyota, C. Ho, N. Ahuja, K. W. Jair, Q. Li, M. O. Toyota, S. B. Baylin and J. P. J. Issa, Cancer Res., 1999, 59, 2307–2312. 3 K. D. Robertson, Nat. Rev. Genet., 2005, 6, 597–610. 4 A. Bird, Genes Dev., 2002, 16, 6–21. 5 M. G. Zulueta, C. M. Bender, A. S. Yang, T. Nguyen, R. W. Beart, J. M. V. Tornout and P. A. Jones, Cancer Res., 1995, 55, 4531–4535. 6 C. I. Stains, J. L. Furman, D. J. Segal and I. Ghosh, J. Am. Chem. Soc., 2006, 128, 9761–9765. 7 D. Macleod, J. Charlton, J. Mullins and A. P. Bird, Genes Dev., 1994, 8, 2282–2292. 8 J. Frigola, J. Song, C. Stirzaker, R. A. Hinshelwood, M. A. Peinado and S. J. Clark, Nat. Genet., 2006, 38, 540–549. 9 A. P. Bird and A. P. Wolffe, Cell, 1999, 99, 451–454. 10 D. Kato, K. Goto, S. I. Fujii, A. Takatsu, S. Hirono and O. Niwa, Anal. Chem., 2011, 83, 7595–7599. 11 F. D. Feng, Y. L. Tang, F. He, M. H. Yu, X. R. Duan, S. Wang, Y. L. Li and D. B. Zhu, Adv. Mater., 2007, 19, 3490–3495. 12 L. Ma, M. Su, T. Li and Z. X. Wang, Analyst, 2014, 139, 3537– 3540. 13 M. Ehrich, S. Zoll, S. Sur and D. V. D. Boom, Nucleic Acids Res., 2007, 35, e29. 14 S. R. Corrie, P. Sova, Q. H. Feng, T. Blair, N. B. Kiviat and M. Trau, Analyst, 2011, 136, 688–691. 15 R. Kurita and O. Niwa, Anal. Chem., 2012, 84, 7533–7538. 16 C. C. Ge, Z. Y. Fang, J. H. Chen, J. Liu, X. W. Lu and L. W. Zeng, Analyst, 2012, 137, 2032–2035. 17 J. Wang, Z. Q. Zhu and H. W. Ma, Anal. Chem., 2013, 85, 2096–2101. 18 A. Okamoto, Org. Biomol. Chem., 2009, 7, 21–26. 19 J. Y. Zhang, B. L. Xing, J. Z. Song, F. Zhang, C. Y. Nie, L. Jiao, L. B. Liu, F. T. Lv and S. Wang, Anal. Chem., 2014, 86, 346– 350. 20 P. A. Jones and P. W. Laird, Nat. Genet., 1999, 21, 163–167. 21 F. Chen and Y. X. Zhao, Analyst, 2013, 138, 284–289. 22 Y. Q. Lv, Q. W. Xue, X. H. Gu, S. Q. Zhang and J. F. Liu, Analyst, 2014, 139, 2583–2588. 23 X. L. Zuo, F. Xia, Y. Xiao and K. W. Plaxco, J. Am. Chem. Soc., 2010, 132, 1816–1818. 24 W. Li, P. Wu, H. Zhang and C. X. Cai, Anal. Chem., 2012, 84, 7583–7590. 25 I. I. Timcheva, V. A. Maximova, T. G. Deligeorgiev, N. I. Gadjev, R. W. Sabnis and I. G. Ivanov, FEBS Lett., 1997, 405, 141–144. 26 T. L. Netzel, K. Nasi, M. Zhao, J. R. Lenhard and I. Johnson, J. Phys. Chem., 1995, 99, 17936–17947. 27 M. Fatemi, M. M. Pao, S. Jeong, E. N. G. Yam, G. Egger, D. J. Weisenberger and P. A. Jones, Nucleic Acids Res., 2005, 33, e176. 28 H. Jaganathan, P. F. Wang, J. Klein, A. C. Lossie and A. Ivanisevic, Analyst, 2011, 136, 2441–2445. 29 S. Bi, T. T. Zhao, B. Y. Luo and J. J. Zhu, Chem. Commun., 2013, 49, 6906–6908. 30 Y. X. Zhao, F. Chen, M. L. Lin and C. H. Fan, Biosens. Bioelectron., 2014, 54, 565–570. 31 K. Kawai, Y. S. Li, M. F. Song and H. Kasai, Bioorg. Med. Chem. Lett., 2010, 20, 260–265.
This journal is © The Royal Society of Chemistry 2014
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
PAPER
Cite this: DOI: 10.1039/c4an01359j
View Journal
Label-free fluorescence detection of DNA methylation and methyltransferase activity based on restriction endonuclease HpaII and exonuclease III Chunyan Gao, Henan Li, Yuanjian Liu, Wei Wei,* Yuanjian Zhang and Songqin Liu Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL1 with a detection limit of 0.16 U mL1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical
Received 24th July 2014 Accepted 24th September 2014
triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no
DOI: 10.1039/c4an01359j
obvious effects on the assay performance, indicating that the method has great potential for further
www.rsc.org/analyst
application in complex samples.
Introduction DNA methylation plays an important role in the regulation of gene transcription, eukaryote development, and cellular processes as well as the pathogenesis of various human diseases such as cancers.1,2 Especially, methylation of cytosine (C) in the specic DNA sequence is an important epigenetic modication,3,4 which has frequently occurred at the carbon 5 position of C in CpG islands (50 -CG-30 ).5–7 In normal cells, most CpG islands spanning the promoter regions are unmethylated, so their downstream genes are active. However, the CpG islands in cancer cells are methylated, and their downstream genes are silenced.8 Abnormal methylation in CpG islands can result in the change of normal cellular functions and phenotypes,9 and has been considered as a potential biomarker of diseases. Thus, a simple system is needed to recognize methylated specic DNA
Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Chemistry and Chemical Engineering, Southeast University, Jiangning District, Nanjing, 211189, P.R. China. E-mail: [email protected]; Fax: +86-25-5209061; Tel: +86-25-52090613
This journal is © The Royal Society of Chemistry 2014
sequences and be applied to early diseases diagnostics and determine cancer types. The conventional sequencing technique cannot be applied to gene-specic methylation detection, owing to methylcytosine (mC) and C displaying identical Watson–Crick base-pair behavior.10 Currently, the methods for detection of DNA methylation are mainly based on distinguishing between mC and C in DNA, including methylation-sensitivity restriction enzyme digestion,11,12 bisulte treatment,13,14 methylation antibody recognition,15,16 and various polymerase chain reaction (PCR) techniques.17 The assay methods based on bisulte and restriction enzymes are widely used to distinguish mC and C in DNA. In the bisulte-based assay, C is deaminated and then converted to uracil (U) through the treatment of bisulte, whereas mC remains almost unchanged due to the extremely low reactivity. The bisulte-based method is currently considered a gold standard assay, yet suffers signicantly from complicated operations and high equipment cost.18 In the restriction enzyme assay, the restriction enzymes sensitively recognize the methylation position and selectively catalyze the scission of the specic DNA sequence. When the C in the specic sequence is methylated, the cleavage is inhibited.12
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
Recently, Wang and his co-workers reported that a methylationsensitive restriction enzyme was used to digest the genic DNA. Only methylated DNA can be amplied by PCR and the methylation level was detected through uorescence resonance energy transfer (FRET) between the cationic conjugated polymer (CCP) and uorescein labeled dNTP of DNA.19 This can provide an economical method to identify the specic site of methylation in DNA and survey the extent of DNA methylation. Moreover, analyzing the MTase activity is also important in the regions of clinical diagnostics because DNA MTase catalyzes the covalent addition of the methyl group to C in DNA in the formation of mC.20,21 Therefore, a fast, cheap, simple, and selective method for detection of DNA methylation and M.SssI MTase activity is imperative. Exonuclease III (Exo III) is a kind of exonuclease that has high exodeoxyribonuclease activity for dsDNA to catalyze the stepwise removal of mononucleotides from the 30 to 50 terminus and limited activity on 30 -overhang ends of dsDNA or ssDNA.22 More recently, Exo III has been extensively used to detect target DNA sensitively through signal amplication. For example, Xiao and co-workers have designed an amplied DNA detection method employing a stem-loop DNA molecular beacon as the signal probe through the cleavage function of exonuclease III on dsDNA.23 Here, the cleavage function of exonuclease III combined with endonuclease HpaII was applied to detect DNA methylation and M.SssI MTase activity. Based on the obvious different uorescence properties of TO in the presence of ssDNA or dsDNA, the method for detection of DNA methylation and M.SssI MTase activity is designed (Scheme 1). The probe and target DNA hybridize to form dsDNA with two 30 protruding termini, which are resistant to cleavage by Exo III. HpaII endonuclease is used to identify the 50 -CCGG-30 sequences and cleave them at the desired position (bottom in Scheme 1), then Exo III could identify the recessed 30 -hydroxyl termini of dsDNA and catalyze the stepwise removal of mononucleotides from this position. The uorescence intensity of the added TO is weak because only ssDNA is contained in the solution (Scheme 1a). If dsDNA was methylated by M.SssI MTase, the function of HpaII was
Paper
inhibited because the cleavage is blocked by CpG methylation, as a result, Exo III cannot work and the dsDNA maintains its integrity. The added TO intercalated into its double strand and showed strong uorescence intensity (Scheme 1b). The uorescence intensity of TO has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL1 with a detection limit of 0.16 U mL1. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These demonstrated that this sensor used for bio-monitoring of site specic DNA methylation and sensitive detection of M.SssI activity is simple, fast and cheap.
Experimental section Chemicals S-Adenosylmethionine (SAM), CpG methyltransferase (M.SssI MTase), restriction endonucleases HpaII and MspI (HpaII) were supplied by Thermo Scientic (USA). Exonuclease III (Exo III) was purchased from TAKARA biotechnology Co., Ltd. (Dalian, China) and used without further purication. Human serum was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Dimethylsulfoxide (DMSO) was purchased from Aladdin reagent Co., Ltd (Shanghai, China) and acetaldehyde (CH3CHO) was obtained from Shanghai Lingfeng Chemical Reagent Co., Ltd (Shanghai, China). Tris(hydroxymethyl)aminomethane (Tris), HCl, NaCl, hydrogen peroxide (30% in water) (H2O2), ascorbic acid, ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA-2Na), and MgCl2$6H2O were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). DL-dithiothreitol (DTT), bovine serum (BSA) and thiazole orange (TO) were of analytical grade and obtained from Sigma-Aldrich. The stock solution of 20 mM TO was prepared in dimethylsulfoxide and diluted with deionized water to a desired concentration. All oligonucleotides were obtained from Shanghai Sangon biotechnology Co., Ltd (Shanghai China). Then, the sequences include single-stranded 32 mer probe DNA (S1: 50 -TCC TGG GAG AGA CCG GCG CAC AGA GGT TAT TA-30 ), a fragment as part of p53 (S2: 50 -CCT CTG TGC GCC GGT CTC TCC CAG GAC AGG CA-30 , target DNA), one base-mismatched target DNA (S3: 50 -CCT CTG TGC GCT GGT CTC TCC CAG GAC AGG CA-30 ), and unrelated DNA (50 -AG ACA GAG GAA AGA AAT TCT CGA AAA ATT ATC-30 ). All solutions of the experiments were prepared with double distilled water. Apparatus All uorescence spectra were obtained using a Fluoromax 4 spectrouorometer (Horiba, Japan), and recorded upon 480 nm excitation with 0.1 s integration time and a 5 nm integration step at room temperature. Hybridization of S1 with target DNA
Scheme 1 Schematic illustration of the principle of label-free fluorescence detection of site-specific DNA methylation and M.SssI MTase activity based on HpaII and Exo III.
Analyst
100 nM probe DNA (S1) and 100 nM target DNA (S2) were incubated in 50 mM Tris–HCl buffer solution (pH 7.9) including 100 mM NaCl and 5 mM MgCl2 at 70 C for 10 min. Then, the solution was cooled down to room temperature. Aer a specic incubation period, TO was added to the solution. Next, the
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
solution was allowed to stand at room temperature for 1 hour, and the uorescence spectra were recorded.
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Methylation of CpG and cleavage of HpaII endonuclease and Exo III The methylation of the S1 (100 nM)/S2 (100 nM) hybrid was performed at 37 C for 1 h in 50 mM Tris–HCl buffer (pH 7.9) containing 50 mM SAM, 100 mM NaCl, 5 mM MgCl2, 0.1 mg mL1 BSA, 1 mM DTT and various concentrations of M.SssI MTase (from 0 to 16 U mL1). Aer methylation, 10 U HpaII and 25 U Exo III were added to the above solution, aerward, cleavage was carried out at 37 C for 2.5 h. Next, 2.5 mM TO was added, the mixture was allowed to stand for 1 h, and the uorescence spectra were recorded without further purication at room temperature. 90 nM unrelated DNA was added into the above S1/S2 hybrid to study their interference with the method. Methylation of cytosine by a hydroxyl radical triggered by DMSO and CH3CHO 100 nM S2 was rst treated with a chemical reagent (100 mM DMSO or CH3CHO in the presence of Fenton reagent (25.0 mM FeSO4, 98.0 mM H2O2, 53.5 mM ascorbic acid, and 15.0 mM EDTA2Na)) through vigorous shaking at room temperature for 3 h, and then it was separated by using Millipore's Amicon Ultra-0.5 centrifugal lter device to remove excessive chemical reagent. The reaction was performed under a N2 atmosphere and the solution is 50 mM Tris–HCl (pH 7.3) containing 100 mM NaCl. Aer that, S1 hybridized with treated S2 in 50 mM Tris–HCl (pH 7.9) containing 100 mM NaCl and 5 mM MgCl2, which was incubated at 70 C for 10 min, and then the solution was cooled down to room temperature. Then, 10 U HpaII and 25 U Exo III were added and cleavage was performed at 37 C for 2.5 h. Finally, 2.5 mM TO was added, the mixture was allowed to stand for 1 h and the uorescence spectra were recorded without further purication at room temperature. The total reaction volume was 500 mL.
Analyst
(S2) and its complementary sequences were designed as probe DNA (S1). TO in an aqueous solution emits almost no uorescence (Fig. 1, curve a). In the presence of probe DNA, TO is adsorbed onto ssDNA through electrostatic attraction, the uorescence of TO has a weak signal (Fig. 1, curve b). In the presence of target DNA, the hybrid of S1/S2 was formed, and TO intercalated into the minor groove of the hybrid by electrostatic interactions. The structure of TO tends to be planar due to the steric effect,25,26 resulting in obvious enhancement of the uorescence signal (Fig. 1, curve c). The uorescence signal is related to the TO concentration, and the optimization of TO concentration is shown in Fig. 2. Both of the uorescence intensities of TO–S1 (Fig. 2, curve a) and TO–S1/S2 (Fig. 2, curve b) increased with the increasing TO concentration. The change in uorescence signal (Fb Fa) also increased along with the increasing TO concentration, tending to balance at 2.5 mM. In order to obtain the highest sensitivity, 2.5 mM was chosen as the optimum TO concentration. The function of HpaII and Exo III and the optimization of their concentration Aer the addition of HpaII into the S1/S2 hybrid solution, the uorescence intensity of TO has no obvious change, indicating that the two parts of the S1/S2 hybrid cleaved by HpaII still retained their duplex form (Fig. 1, curve d). Upon the addition of
Detection M.SssI activity in human serum samples 500 nM S1/S2 hybrid was added to 100 mL of the human serum sample. Then, DNA was extracted from the above sample with a nucleic acid automatic extraction system (Applied Biosystems, Thermo Fisher Scientic Oy, USA) and was diluted 5 times with 50 mM Tris–HCl buffer before use. In addition, 100 nM S1/S2 hybrid added to a 10-fold diluted human serum sample without extraction was used as a control.
Fig. 1 Fluorescence spectra (left panel) and histogram (right panel) of 2.5 mM TO (a), TO in the presence of S1 (b), S1/S2 (c), S1/S2 and HpaII (d), S1/S2, HpaII and Exo III (e), S1/S2 and Exo III (f), S1/S2, M.SssI, HpaII and Exo III (g) and S1/S2, M.SssI, MspI and Exo III (h).
Results and discussion Fluorescence properties of TO and its concentration optimization p53 has proved to be a tumor-suppressor gene. The methylation of the region of p53 plays a key role in the development of at least 60% of tumors of the colon, breast, lung, ovaries, cervix, adrenal cortex, bone, and bladder and at least 30% of brain tumors.24 So, a part of sequences of the p53 gene containing a specic methylation site (50 -CCGG-30 ) was chosen as target DNA
This journal is © The Royal Society of Chemistry 2014
Fig. 2 Fluorescence intensity of various concentrations of TO in the presence of S1 (a), S1/S2 (b) and Fb Fa (c). Fa is the fluorescence intensity of TO–S1 and Fb is the fluorescence intensity of the TO–S1/S2.
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
both Exo III and HpaII into the S1/S2 hybrid solution, the uorescence intensity of TO decreased nearly to that in the ssDNA (S1), which proved the digestion function of Exo III (Fig. 1, curve e). When only Exo III was added to the S1/S2 hybrid solution (Fig. 1, curve f), the TO intensity did not change, indicating that the cleavage function of HpaII is efficient and necessary. The effects of HpaII and Exo III concentration and reaction time were investigated. The uorescence intensity of TO decreased with the increasing concentration of HpaII in the range of 0–20 U mL1. Further increase of HpaII concentration caused little enhancement of the uorescence signal in the range of 20–60 U mL1. Therefore, 20 U mL1 HpaII was chosen as the optimum concentration for all measurements (Fig. 3A). In the presence of 20 U mL1 HpaII, the uorescence intensity of TO decreased with the increasing concentration of Exo III and tended to a plateau at 50 U mL1, indicating that the maximum degradation efficiency was obtained. Therefore, 50 U mL1 was chosen in the following experiments (Fig. 3B). The reaction time of HpaII and Exo III also affected the uorescence signal, which reduced sufficiently with increasing reaction time and had a tendency to a constant value at 140 min (Fig. 3C). As a result, 150 min was chosen as the optimal reaction time. Detection of the target DNA and its selectivity The quantitative detection of DNA by the proposed uorescence sensor was investigated under the optimized conditions. As
Paper
shown in Fig. 4A, the uorescence intensity at 528 nm increased with the increasing concentration of target DNA from 0 nM to 140 nM. Fig. 4B depicts that the uorescence signal increased with the concentration of target DNA from 0 to 100 nM, and reached a plateau in the range of 100–140 nM. The linear correlation was obtained in the concentration range of 0.6 nM to 80 nM and the linear correlation was 0.992 (inset in Fig. 4B). The detection limit of 0.1 nM was obtained in terms of 3 times deviation of the blank sample. One base mismatched target DNA (S3) was used to evaluate the selectivity of the method. Compared with the uorescence intensity of TO–S1/S2 (Fig. 5a), the intensity of TO–S1/S3 (Fig. 5b) decreased only about 10%, indicating that TO has poor selectivity for the one base mismatched target DNA. When the S1/S2 hybrid was treated with 10 U HpaII and 25 U Exo III for 2.5 h at 37 C, the uorescence response of TO decreased by about 60% (Fig. 5c) compared with the uorescence intensity of TO–S1/S2. However, the intensity of TO in the presence of S1/S3 that was treated with 10 U HpaII and 25 U Exo III changed little compared with that not treated with HpaII and Exo III (Fig. 5d). This demonstrated that HpaII and Exo III do not work in the presence of one base mismatched target DNA, indicating that the proposed method based on TO (signal molecule), HpaII (cleavage of the ds DNA into two parts) and Exo III (degradation of dsDNA to ssDNA) has good selectivity to differentiate one base mismatched DNA from intact target DNA.
Detection of M. SssI activity
Fig. 3 The fluorescence intensity of TO as a function of the concentration of (A) HpaII, (B) Exo III, and (C) the reaction time of HpaII and Exo III.
Fig. 4 (A) Corresponding fluorescence spectra of TO in the presence of S1 and 0 nM, 0.6 nM, 4 nM, 10 nM, 20 nM, 40 nM, 60 nM, 80 nM, 100 nM, 120 nM and 140 nM target DNA (a–k), and (B) the standard curve for the relationship between the fluorescence signal and the concentration of target DNA, the inset shows a linear relationship between the fluorescence signal and the concentration of target DNA.
Analyst
It is known that in the presence of M.SssI MTase and a methyl group donor, S-adenosyl-L-methionine (SAM),27 the second cytosine at the sequence of 50 -CCGG-30 can be methylated to 50 -CmCGG-30 . The methylated cytosine prevents dsDNA from cutting by HpaII (ref. 28) and degrading by Exo III. In the presence of M.SssI MTase, the S1/S2 hybrid was methylated at the specic site of 50 -CG-30 , the treatment of HpaII and Exo III did not change the uorescence of the TO signal (Fig. 1, curve g). However, when the methylated S1/S2 was treated with restriction endonuclease MspI and Exo III, the uorescence signal of TO decreased (Fig. 1, curve h), because MspI is not sensitive to methylation, suggesting that isoschizomer MspI does not work in the experiments. Thus, the activity of M.SssI
Fig. 5 Fluorescence spectra (left panel) and histogram (right panel) of TO–S1/S2 (a), TO–S1/S3 (b), TO–S1/S2 (c) and TO–S1/S3 (d) incubated with HpaII and Exo III at 37 C for 2.5 h in 50 mM Tris–HCl (pH 7.9).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Paper
can be detected by comparing the uorescence of TO–S1/S2 in the presence of various concentrations of M.SssI. The methylation level was closely dependent on the reaction time of M.SssI, we rstly evaluate the effect of methylation time in the methylation process. The S1/S2 hybrid was methylated for various times under the function of M.SssI MTase, then the uorescence signal was recorded aer the cleavage of methylated hybrid by HpaII and Exo III (Fig. 6). The uorescence signal increased rapidly in the range of 0–40 min and tended to a plateau in the range of 40–120 min. Therefore, 1 h was chosen as the optimal time for full reaction. The S1/S2 hybrid was methylated with various concentrations of M.SssI MTase from 0 U mL1 to 16 U mL1 for 1 h, and then incubated with HpaII and Exo III. Aer reaction for 2.5 h, 2.5 mM TO was added to the solution. The intensity of uorescence signals increased with the increasing concentration of M.SssI MTase (Fig. 7A) because more S1/S2 hybrid is methylated and the cleavage and degradation functions of HpaII and Exo III are prevented at higher concentrations of M.SssI MTase. Fig. 7B reveals that the uorescence signals increased with the increasing concentration of M.SssI MTase from 0 to 12 U mL1, and reached a constant value at 12 U mL1. A linear correlation
Analyst
was obtained in the concentration range of 1 U mL1 to 10 U mL1 and the correlation coefficient was 0.997 (inset in Fig. 7B). The detection limit of 0.16 U mL1 was obtained in terms of 3 times deviation of the blank sample, which is lower than that of the chemiluminescence (CL) method (0.52 U mL1)29 and the label-free colorimetric method (0.4 U mL1).30 Thus, this proposed method can be applied to sensitive detection of MTase activity. Detection of cytosine methylation induced by a hydroxyl radical triggered by DMSO and CH3CHO The method has also been used to detect the methylation of DNA cytosine by DMSO and CH3CHO in the presence of Fenton reagent which provided a hydroxyl radical. The target DNA was treated with DMSO or CH3CHO in the presence Fenton reagent at room temperature for 3 h, then the mixture was separated through a centrifugal lter device (molecular weight 3 kD, centrifugal speed 14 000 rcf, 15 min). Aer ultraltration, S1 hybridized with treated S2, and then 10 U HpaII and 25 U Exo III were added for incubation. Fig. 8 shows that the uorescence intensity of HpaII and Exo III cut S1/S2 hybrid (Fig. 8, curve b) was much lower than that of the S1/S2 hybrid (Fig. 8, curve a). When S2 was treated with CH3CHO (Fig. 8A, curve c) or DMSO (Fig. 8A, curve d) in the presence of Fenton reagent, the uorescence intensity was stronger than that of intact S1/S2. These results indicated that these two chemical reagents methylated the target DNA, as a result, the functions of HpaII and Exo III are prevented. This was in agreement with the reported results that the methylation of cytosine occurred through methyl radicals produced by DMSO and CH3CHO in the presence of Fenton reagent.31 Therefore, this method can be used to detect the DNA methylation induced by chemical reagents. Application of the method in human serum samples
The fluorescence signal of TO was dependent on the methylation time with M.SssI. Fig. 6
Fig. 7 (A) The effect of M.SssI concentration on the fluorescence response of TO. The concentration of M.SssI is (a) 0, (b) 1, (c) 2, (d) 3, (e) 4, (f) 5, (g) 6, (h) 8, (i) 9, (j) 10, (k) 12, and (l) 16 U mL1, respectively. Before the fluorescence spectra were recorded, the S1/S2 hybrid was methylated by M.SssI and 50 mM SAM for 1 h, and then incubated with HpaII and Exo III for 2.5 h, finally TO was added. (B) The plot of the fluorescence signal vs. M.SssI concentration, the inset: the linear relationship between the fluorescence signal and the concentration of the M.SssI concentration.
This journal is © The Royal Society of Chemistry 2014
The application of the method in complex biological matrices is another challenging factor to evaluate the performance of the sensor. 90 nM unrelated sequences were used as interference materials. As depicted in Fig. 9A, it was observed that 90 nM unrelated sequence has no obvious effect on the detection, however, the uorescence intensity for methylated DNA (c) and intact DNA (b) showed obvious differences. 10% diluted human
Fig. 8 Fluorescence spectra (left panel) and histogram (right panel) of TO–S1/S2 (a), TO–S1/S2 containing HpaII and Exo III (b), S2 was treated with Fenton reagent including CH3CHO (c), and Fenton reagent including DMSO (d) following the same procedures as that in (b).
Analyst
View Article Online
Published on 24 September 2014. Downloaded by Northeastern University on 02/11/2014 03:29:53.
Analyst
Fig. 9 (A) Results obtained from (a) S1/S2, (b) S1/S2 with HpaII and Exo III, (c) S1/S2 with 7 U M.SssI MTase, HpaII and Exo III in the 50 mM Tris– HCl buffer solution (black column) and the 50 mM Tris–HCl buffer solution containing 90 nM unrelated DNA (red column). (B) The comparison of signals of (a) S1/S2, (b) S1/S2 with HpaII and Exo III, (c) S1/S2 with 7 U M.SssI MTase, HpaII and Exo III in 10% diluted human serum samples (red column), 20% diluted DNA extraction of human serum samples (blue column) and 50 mM Tris–HCl buffer solution (black column).
serum samples (red column) and 20% diluted DNA extraction samples (blue column) were investigated, respectively. Fig. 9B shows that the signal ratio of methylated DNA (b) to intact DNA (c) for black, red and blue columns were 2.5, 1.8 and 3, which indicated that the method can be used to differentiate the methylated DNA from intact DNA in human serum samples. It was also obvious that aer the DNA extraction, the assay of the human serum sample is more sensitive.
Conclusions To sum up, we have presented a label-free uorescence method to detect DNA methylation and M.SssI activity based on HpaII and Exo III. The method operates via directly detecting the TO uorescence intensity, provides a rapid, simple, homogeneous phase and site-specic methylation assay, and requires no bisulte change, PCR amplication, label probe and separation. The method also has good selectivity to differentiate one base mismatched target DNA from intact DNA. As the epigenetic basis of cancerous transformation remains to be discovered, the demand for effective and inexpensive strategies to detect human methyltransferase activity will continue to grow. Human serum samples have no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
Acknowledgements The project is supported by the National Natural Science Foundation of China (Grant no. 21175021, 21205014, and 21475020), and the Natural Science Foundation of Jiangsu province (BK2012734).
Notes and references 1 K. D. Robertson and A. P. Wolffe, Nat. Rev. Genet., 2000, 1, 11–19.
Analyst
Paper
2 M. Toyota, C. Ho, N. Ahuja, K. W. Jair, Q. Li, M. O. Toyota, S. B. Baylin and J. P. J. Issa, Cancer Res., 1999, 59, 2307–2312. 3 K. D. Robertson, Nat. Rev. Genet., 2005, 6, 597–610. 4 A. Bird, Genes Dev., 2002, 16, 6–21. 5 M. G. Zulueta, C. M. Bender, A. S. Yang, T. Nguyen, R. W. Beart, J. M. V. Tornout and P. A. Jones, Cancer Res., 1995, 55, 4531–4535. 6 C. I. Stains, J. L. Furman, D. J. Segal and I. Ghosh, J. Am. Chem. Soc., 2006, 128, 9761–9765. 7 D. Macleod, J. Charlton, J. Mullins and A. P. Bird, Genes Dev., 1994, 8, 2282–2292. 8 J. Frigola, J. Song, C. Stirzaker, R. A. Hinshelwood, M. A. Peinado and S. J. Clark, Nat. Genet., 2006, 38, 540–549. 9 A. P. Bird and A. P. Wolffe, Cell, 1999, 99, 451–454. 10 D. Kato, K. Goto, S. I. Fujii, A. Takatsu, S. Hirono and O. Niwa, Anal. Chem., 2011, 83, 7595–7599. 11 F. D. Feng, Y. L. Tang, F. He, M. H. Yu, X. R. Duan, S. Wang, Y. L. Li and D. B. Zhu, Adv. Mater., 2007, 19, 3490–3495. 12 L. Ma, M. Su, T. Li and Z. X. Wang, Analyst, 2014, 139, 3537– 3540. 13 M. Ehrich, S. Zoll, S. Sur and D. V. D. Boom, Nucleic Acids Res., 2007, 35, e29. 14 S. R. Corrie, P. Sova, Q. H. Feng, T. Blair, N. B. Kiviat and M. Trau, Analyst, 2011, 136, 688–691. 15 R. Kurita and O. Niwa, Anal. Chem., 2012, 84, 7533–7538. 16 C. C. Ge, Z. Y. Fang, J. H. Chen, J. Liu, X. W. Lu and L. W. Zeng, Analyst, 2012, 137, 2032–2035. 17 J. Wang, Z. Q. Zhu and H. W. Ma, Anal. Chem., 2013, 85, 2096–2101. 18 A. Okamoto, Org. Biomol. Chem., 2009, 7, 21–26. 19 J. Y. Zhang, B. L. Xing, J. Z. Song, F. Zhang, C. Y. Nie, L. Jiao, L. B. Liu, F. T. Lv and S. Wang, Anal. Chem., 2014, 86, 346– 350. 20 P. A. Jones and P. W. Laird, Nat. Genet., 1999, 21, 163–167. 21 F. Chen and Y. X. Zhao, Analyst, 2013, 138, 284–289. 22 Y. Q. Lv, Q. W. Xue, X. H. Gu, S. Q. Zhang and J. F. Liu, Analyst, 2014, 139, 2583–2588. 23 X. L. Zuo, F. Xia, Y. Xiao and K. W. Plaxco, J. Am. Chem. Soc., 2010, 132, 1816–1818. 24 W. Li, P. Wu, H. Zhang and C. X. Cai, Anal. Chem., 2012, 84, 7583–7590. 25 I. I. Timcheva, V. A. Maximova, T. G. Deligeorgiev, N. I. Gadjev, R. W. Sabnis and I. G. Ivanov, FEBS Lett., 1997, 405, 141–144. 26 T. L. Netzel, K. Nasi, M. Zhao, J. R. Lenhard and I. Johnson, J. Phys. Chem., 1995, 99, 17936–17947. 27 M. Fatemi, M. M. Pao, S. Jeong, E. N. G. Yam, G. Egger, D. J. Weisenberger and P. A. Jones, Nucleic Acids Res., 2005, 33, e176. 28 H. Jaganathan, P. F. Wang, J. Klein, A. C. Lossie and A. Ivanisevic, Analyst, 2011, 136, 2441–2445. 29 S. Bi, T. T. Zhao, B. Y. Luo and J. J. Zhu, Chem. Commun., 2013, 49, 6906–6908. 30 Y. X. Zhao, F. Chen, M. L. Lin and C. H. Fan, Biosens. Bioelectron., 2014, 54, 565–570. 31 K. Kawai, Y. S. Li, M. F. Song and H. Kasai, Bioorg. Med. Chem. Lett., 2010, 20, 260–265.
This journal is © The Royal Society of Chemistry 2014
