Novel Method to Detect DNA Methylation Using Gold Nanoparticles

Dec 2, 2009 - DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as ...
0 downloads 0 Views 764KB Size
Download PDF
Anal. Chem. 2010, 82, 229–233

Novel Method to Detect DNA Methylation Using Gold Nanoparticles Coupled with Enzyme-Linkage Reactions Tao Liu,† Jing Zhao,† Dongmei Zhang,† and Genxi Li*,†,‡ Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P. R. China, and Laboratory of Biosensing Technology, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China DNA methylation, catalyzed by methylases, plays a critical role in many biological processes, and methylases have been regarded as promising targets for antimicrobial drugs. In this paper, we propose a simple and sensitive colorimetric assay method to detect the activity of methylases so as to monitor DNA methylation using DNAmodified gold nanoparticles (AuNPs) coupled with enzymelinkage reactions. The duplex DNA molecules modified on the surface of AuNPs are first methylated by DNA adenine methylation (Dam) methyltransferase (MTase) and then cut by methylation-sensitive restriction endonuclease Dpn I. Removal of duplex from the AuNP surfaces by the methylation/cleavage process will destabilize the nanoparticles, resulting in aggregation of AuNPs and a red-to-blue color change. Consequently, the enzyme activity of Dam MTase can be assayed and DNA methylation can be detected. Furthermore, this study may provide a sensitive platform to screen inhibitors for Dam MTase. DNA methylation, a critical process existing in both prokaryotes and eukaryotes, is achieved by the catalysis of DNA methyltransferases (MTase). DNA MTases specifically recognize the short palindromic binding sequences and catalyze the transfer of a methyl group from S-adenosyl-L-methionine to the target cytosine or adenine. Aberrant DNA methylation induced by microbe remodels the structure of DNA, influences the interaction between DNA and protein, and alters gene expression, which may result in tumor occurrence and tumor growth.1-4 On the other hand, MTases have been regarded as a novel family of pharmacological targets for the treatment of tumors. In addition, the inhibition of MTases may provide a broad spectrum of antimicrobial applications.5,6 Therefore, * Corresponding author. E-mail: [email protected]. † Nanjing University. ‡ Shanghai University. (1) Heithoff, D. M.; Sinsheimer, R. L.; Low, D. A.; Mahan, M. J. Science 1999, 284, 967–970. (2) Robertson, K. D.; Wolffe, A. P. Nat. Rev. Genet. 2000, 1, 11–19. (3) Jeltsch, A. Chem. Biol. Chem. 2002, 3, 274–293. (4) Shames, D. S.; Minna, J. D.; Gazdar, A. F. Curr. Mol. Med. 2007, 7, 85– 102. (5) Brueckner, B.; Lyko, F. Trends Pharmacol. Sci. 2004, 25, 551–554. (6) Mashhoon, N.; Pruss, C.; Carroll, M.; Johnson, P. H.; Reich, N. O. J. Biomol. Screening 2006, 11, 497–510. 10.1021/ac902198v  2010 American Chemical Society Published on Web 12/02/2009

detection of DNA methylation and assay of MTase activities have received more and more research interests. Traditional methods used for the detection of DNA methylation include methylation-specific polymerase chain reaction (PCR)7 and real-time PCR-based methylation-specific PCR (MSP).8 However, these approaches lack the sensitivity for direct screening of challenging samples where the DNA from the tumor cells is minimal, thereby requiring a nested PCR approach.9 Recently, methylation-specific quantum dot fluorescence resonance energy transfer10 (MS-qFRET) and Methyl-BEAMing11 (beads, emulsion, amplification, and magnetics) technology facilitate a straightforward approach for detection of low-abundance methylated DNA. Meanwhile, several methods have been developed to assay DNA MTase activities such as gel electrophoresis, radioactive labeling, and high-performance liquid chromatography (HPLC). However, these techniques are discontinuous, time-consuming, and usually require radio-labeled substrates.12-14 Hence, a new method based on fluorescence using hairpin fluorescent molecular beacon was recently reported, which may overcome these limitations,15,16 but the fluorescence-based assay technique needs double labeled DNA probes and is facilely affected by external nonspecific events. Therefore, development of sensitive, simple, and economical methods for DNA methylation detection and DNA MTase activity assays is highly required. DNA-modified gold nanoparticles (AuNPs), with unique sizeand distance-dependent optical properties, have attracted consider(7) Herman, J. G.; Graff, J. R.; Myo ¨ha¨nen, S.; Nelkin, B. D.; Baylin, S. B. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 9821–9826. (8) Lo, Y. M.; Wong, I. H.; Zhang, J.; Tein, M. S.; Ng, M. H.; Hjelm, N. M. Cancer Res. 1999, 59, 3899–3903. (9) Brandes, J. C.; van Engeland, M.; Wouters, K. A.; Weijenberg, M. P.; Herman, J. G. Carcinogenesis 2005, 26, 1152–1156. (10) Bailey, V. J.; Easwaran, H.; Zhang, Y.; Griffiths, E.; Belinsky, S. A.; Herman, J. G.; Baylin, S. B.; Carraway, H. E.; Wang, T. Genome Res. 2009, 19, 1455– 1461. (11) Li, M.; Chen, W.; Papadopoulos, N.; Goodman, S. N.; Bjerregaard, N. C.; Laurberg, S.; Levin, B.; Juhl, H.; Arber, N.; Moinova, H.; Durkee, K.; Schmidt, K.; Markowitz, S. D.; Vogelstein, B. Nat. Biotechnol. 2009, 27, 858–863. (12) Messer, W.; Noyer-Weidner, M. Cell 1988, 54, 735–737. (13) Bergerat, A.; Guschlbauer, W.; Fazakerley, G. V. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 6394–6397. (14) Jeltsch, A.; Fritz, A.; Alves, J.; Wolfes, H.; Pingoud, A. Anal. Biochem. 1993, 213, 234–240. (15) Li, J.; Yan, H.; Wang, K.; Tan, W.; Zhou, X. Anal. Chem. 2007, 79, 1050– 1056. (16) Tan, W.; Wang, K.; Drake, T. Curr. Opin. Chem. Biol. 2004, 16, 173–176.

Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

229

Scheme 1. Schematic Illustration of the Proposed Strategy for Methylation Detection in This Work

able attention in the fields of biosensors17,18 and nanotechnology.19,20 Although AuNPs functionalized with DNA have been widely employed to fabricate assembly and disassembly of nanostructures via DNA hybridization19-22 and the DNA molecules tethered on the AuNP surfaces have also been known to be manipulated in a desirable manner by enzymatic reactions that are routinely used in molecular biology such as cleavage, ligation, and polymerization,23-29 there have been only a few studies for biosensing purposes based on enzymatic manipulation of DNA-modified AuNPs.30-34 Mirkin and co-workers32 have reported a colorimetric detection for DNase I endonuclease activity. The DNA substrates of DNase I serve as cross-linkers in AuNP aggregates where the substrate strand are cleaved by DNase I, resulting in the dissociation of AuNPs. The similar process has been investigated by Ren and co-workers for assays of different kinds of restriction endonuclease activities, and MTases activity can be detected by this technique,33 which is based upon the process of DNAmodified AuNPs from aggregates to dispersion. In this paper, we present an alternative strategy for the assay of MTase activity and the detection of DNA methylation based on the aggregation of AuNPs from a well-dispersed state.34,35 The mechanism of this proposed strategy has been illustrated in Scheme 1. The AuNPs modified with double stranded DNA (dsDNA) are stable at a given salt concentration due to the electrostatic and steric stabilization provided by tethered nega(17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35)

230

Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547–1562. Lu, Y.; Liu, J. Curr. Opin. Biotechnol. 2006, 17, 580–588. Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. Baron, R.; Willner, B.; Willner, I. Chem. Commun. 2007, 323–339. Zheng, J.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A.; Seeman, N. C. Nano Lett. 2006, 6, 1502–1504. Kanaras, A. G.; Wang, Z.; Bates, A. D.; Cosstick, R.; Brust, M. Angew. Chem., Int. Ed. 2003, 42, 191–194. Kanaras, A. G.; Wang, Z.; Hussain, I.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 67–70. Wang, Z.; Kanaras, A. G.; Bates, A. D.; Cosstick, R.; Brust, M. J. Mater. Chem. 2004, 14, 578–580. Kanaras, A. G.; Wang, Z.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 590–594. Nicewarner Pena, S. R.; Raina, S.; Goodrich, G. P.; Fedoroff, N. V.; Keating, C. D. J. Am. Chem. Soc. 2002, 124, 7314–7323. Zhao, W.; Gao, Y.; Kandadai, S. A.; Brook, M. A.; Li, Y. Angew. Chem., Int. Ed. 2006, 45, 2409–2413. Yun, C. S.; Khitrov, G. A.; Vergona, D. E.; Reich, N. O.; Strouse, G. F. J. Am. Chem. Soc. 2002, 124, 7644–7645. Qin, W. J.; Yung, L. Y. L. Langmuir 2005, 21, 11330–11334. Liu, J.; Lu, Y. J. Am. Chem. Soc. 2003, 125, 6642–6643. Liu, J.; Lu, Y. J. Am. Chem. Soc. 2004, 126, 12298–12305. Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem. 2007, 119, 3538–3540. Song, G. T.; Chen, C.; Ren, J. S.; Qu, X. G. ACS Nano 2009, 3, 1183–1189. Wang, J.; Wang, L.; Liu, X.; Liang, Z.; Song, S.; Li, W.; Li, G. X.; Fan, C. H. Adv. Mater. 2007, 19, 3943–3946. Zhao, W.; Lam, J.; Chiuman, W.; Brook, M. A.; Li, Y. F. Small 2008, 4, 810–816.

Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

tively charged DNA polymers.36 Nevertheless, if there is DNA adenine methylation (Dam) MTase in the test solution, the 5′-GA-T-C-3′ recognition sequence on the dsDNA are, thus, recognized and methylated by the enzyme. Subsequently, the methylated dsDNA are specially recognized and cleaved by methylation sensitive restriction endonuclease Dpn I (Dpn I) at the site of 5′-G-Am-T-C-3′.37,38 Loss of protecting tethered duplex due to the coupled methylation/cleavage reactions will destabilize the AuNPs, leading to a rapid aggregation of AuNPs and a red-to-blue color change. Accordingly, the colorimetric change is able to effectively assay the activity of Dam MTase and indicate the methylation of the DNA. In addition, the screening of the inhibitors of MTase can be achieved by employing DNA-modified AuNPs coupled with methylation/cleavage reactions. EXPERIMENTAL SECTION Reagents and Materials. Hydrogen tetrachloroaurate trihydrate (HAuCl4 · 3H2O), S-adenosyl-L-methiolnine (SAM), dithiothreitol (DTT), Tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), sodium citrate, and other salts were obtained from Sigma-Aldrich (St. Louis, MO). Acrylamide, N,N,N′,N′-tetramethylethylenediamine, and ethidum bromide (EB) were supplied from SBS (Beijing, China). The Dam MTase (Escherichia coli) and Dpn I endonuclease were purchased from New England Biolabs Inc. Other chemicals were of analytical grade and were used without further purification. For all the experiments, Milli-Q water (18.0 MΩ) was used, purified by a Milli-Q Plus 185 ultrapure water system (Millipore purification pack). DNA oligonucleotides were synthesized by Sangon (Shanghai, China) and used without further purification. The DNA sequences are as follows:

Preparation of AuNPs. The 13 nm AuNPs were prepared using a standard citrate method.39,40 Briefly, trisodium citrate (25 mL, 38.8 mM) was added to a boiling solution of HAuCl4 (250 mL, 1 mM). Within several minutes, the color of the solution (36) Napper, D. H. Polymeric Stabilization of Colloidal Dispersions; Academic Press: New York, 1983. (37) Garcia-Del Portillo, F.; Pucciarelli, M. G.; Casadesus, J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11578–11583. (38) Geier, G. E.; Modrich, P. J. Biol. Chem. 1979, 254, 1408–1413. (39) Jin, R. C.; Wu, G. S.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643–1654. (40) Liu, J.; Lu, Y. Nat. Protoc. 2006, 1, 246–252.

was changed from pale yellow to deep red. The mixture was allowed to heat under reflux for another 30 min to ensure complete reduction and was slowly cooled down to room temperature. The diameter of such prepared nanoparticles was measured to be ∼13 nm, and the nanoparticle concentration was ∼13 nM. Functionalization of AuNPs. The AuNPs functionalized with DNA were prepared according to the previously reported method.40 Before DNA loading on the AuNP surface, thiolmodified DNA S1 (9 µL, 1 mM) was activated by treatment with TCEP (1 µL, 500 mM acetate buffer, pH 5.2) at room temperature for 1 h. AuNPs were then functionalized by transferring the deprotected DNA S1 into 3 mL of citrate-stabilized AuNPs. The resulting solution was stored in a drawer at room temperature for at least 16 h. A magnetic stirring bar was applied to facilitate the reaction. After that, tris acetate buffer (30 µL, 500 mM, pH 8.2) was added dropwise to the solution making a final Tris acetate concentration of 5 mM. Finally, the solution was incubated for another day at room temperature after NaCl was added, the concentration of which in the solution was 100 mM. The S1-modified AuNPs were purified three times by centrifugation (16 600g for 15 min) in Tris-HCl (10 mM, pH 7.5). The S1DNA modified on the surfaces of AuNPs was then hybridized with S2-DNA as follows: S2 (9 µL, 100 µM) was added to S1-modified AuNP solution (600 µL, 3 nM). The solution was heated to 90 °C, held at this temperature for 10 s, and then slowly cooled to room temperature for 30 min. Excess S2 was removed by centrifugation (16 600g for 15 min) in Tris-HCl (10 mM, pH 7.5). The DNA functionalized AuNP solution was stored in a refrigerator at 4 °C. MCH Treatment. The above prepared DNA functionalized AuNPs were first diluted to 3 nM with Tris-HCl (10 mM, pH 7.5). MCH (0.2 mM, final concentration) was then added to the AuNP solutions. The MCH treatment was carried out at 25 °C for 8 min, and the reaction was halted by three washes with a 3× volume of ethyl acetate, which extracted excess MCH out of the aqueous solution. MCH extraction is crucial for this work since it permits control of the reaction time, which is essential for avoiding complete displacement of the DNA.41 The DNA functionalized AuNPs were then centrifugated and redispersed in 10 mM Tris-HCl (50 mM NaCl, pH 7.5) for further use. Assay of Dam MTase Activity and Methylation. A series of standard Dam MTase solutions were prepared from 10 to 100 units. To protect the activity of Dam MTase, all the standard solutions were prepared under 4 °C and stored under -20 °C. A typical Dam MTase assay solution contained dsDNA-functionalized AuNPs (referred as AuNP/S1-2, 2.5 nM), MgCl2 (10 mM), NaCl (50 mM), Tris-HCl (10 mM, pH 7.5), SAM (80 µM), Dpn I (20 units/mL), and various amounts of Dam MTase. The ratios of absorbance A620/A520 were monitored every 10 s at 37 °C using a UV-vis spectrometer (UV-2450, Shimadzu, Japan). Inhibition of Dam MTase Activity. The inhibition effect could be also quantitatively analyzed using the ratios of absorbance A620/ A520. A typical mixture contained AuNP/S1-2 (3 nM), SAM (80 µM), Dpn I (20 units/mL), 1× reaction buffer, and 1 µM inhibitor. The assay was initiated by adding Dam MTase (8.0 units/mL), and the ratios of absorbance A620/A520 were continu(41) Park, S.; Brown, K. A.; Hamad-Schifferli, K. Nano Lett. 2004, 4, 1925– 1929.

ously measured over a time course of 6 min. To investigate the relationship between the concentration of a specific inhibitor and the inhibition ratio, different concentrations of this inhibitor were added in the test solutions. The control experiments were performed as follows: Dam MTase (8.0 units/mL) was added to a reaction mixture without Dpn I, and the mixture was incubated at 37 °C for 1 h to complete methylation. Then, Dpn I was added, and the ratios of absorbance A620/A520 were recorded as described above. Assay of Methylation by Gel Electrophoresis. Before the gel electrophoresis assay, the 10 µL sample (5 µM hybridized DNA, 10 units of Dam MTase and Dpn I, 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 80 µM SAM, pH 7.5) was incubated at 37 °C for 3 h. The samples were then put on a polyacrylamide gel (20% acrylamide, 19:1, acrylamide/bisacrylamide) to separate the cleaved products from the substrate. The electrophoresis was carried in 1× tris-borate-EDTA (TBE) (pH 8.0) at 200 V constant voltages for 1 h. After EB staining, the gel was scanned using the Image Master VDS-SL (Amersham). RESULTS AND DISCUSSION Optimization of Assay System. According to the SchulzeHardy rule, the stability of AuNPs is strongly influenced by divalent positive ions (such as Mg2+) compared with monovalent ions (such as Na+); thus, a much lower concentration of divalent ions may induce aggregation of AuNPs. Herein, the stabilities of AuNPs modified separately by S1-2 and S1’ toward Mg2+-induced aggregation have been studied by gradually adding 1 M MgCl2 stock solution to the corresponding solution. The freshly prepared S1-2 functionalized AuNPs are shown to endure concentrations of MgCl2 up to 60 mM, while the AuNPs modified with S1’ are only stable at the MgCl2 concentration e20 mM. It should be pointed out that 20 mM MgCl2 is considered to significantly decrease the activity of restriction endonuclease Dpn I.15 Thereby, MCH exchange reaction has been carried out to decrease the salt endurance of the DNA modified AuNPs, because after the ligand exchange reaction to remove some of the chemically bound DNA molecules from the nanoparticle surfaces, the stability of the AuNPs in a salt environment can be decreased to a much lower level. Meanwhile, due to the removal of some DNA molecules from the AuNP surfaces, the accessibility of the enzymes to the DNA molecules can be also greatly enhanced42,43 which will facilitate the methylation and cleavage reaction; thus, the detection sensitivity can be increased. Experimental results reveal that 10 mM Mg2+ is the optimum concentration that offers the highest activity of DNA methylation for our system; thus, we have chosen 10 mM Mg2+ in the following experiments. Figure 1 shows the stabilities of the AuNPs separately functionalized by S1-2 and S1’ toward 10 mM MgCl2 treated with MCH for different times. It can be known that after incubation of more than 8 min, A620/A520 of the AuNPs for the S1-2 case starts to increase, indicating the aggregation of AuNPs; while for the S1’ case the AuNPs become unstable when the incubation time is 3 min. This not only shows the instability of the nanoparticles due to the poor protection of (42) Seferos, D. S.; Prigodich, A. E.; Giljohann, D. A.; Patel, P. C.; Mirkin, C. A. Nano Lett. 2009, 9, 308–311. (43) Qin, W. J.; Lanry-Yung, L. Y. Biomacromolecules 2006, 7, 3047–3051.

Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

231

Figure 1. Salt endurances of AuNP/S1-2 and AuNP/S1’ treated with MCH for different times. MgCl2 concentration: 10 mM.

the DNA molecules on the AuNP surfaces but also reveals that the AuNPs functionalized with S1-2 should be treated with MCH for 8 min in the following experiment. Furthermore, we have performed fluorescent tests to show that the number of DNA molecules tethered on each AuNP has been decreased from ∼90 to ∼35 after an 8 min duration treatment, and the AuNPs after MCH treatment can be still stored for >1 month if there is no Mg2+ in the stock solution. Assay of Dam MTase Activity. As is mentioned above, the removal of DNA strands from the surfaces of nanoparticles can induce destabilization and aggregation of AuNPs driven by van der Waals attraction, since DNA strands are known to serve as electrostatic and steric stabilizers at relatively high salt concentration;35,36 thus, we have proposed a method to assay the activity of Dam MTase. First, if there is Dam MTase in the test solution, the enzyme will catalyze the methylation reaction on the recognition sequence to yield the methylation duplex DNA 5′-G-Am-TC-3′. Then, the cleavage reaction by Dpn I restriction endonuclease is sequentially initiated. Owing to the loss of protection duplex on the surfaces of the nanoparticles, AuNPs begin to aggregate, and a red-to-blue color change can be observed in a few minutes. Certainly, more precise measurements can also be performed ascribing to a red shift and broadening of surface plasmon band of AuNP in the UV-vis spectrum (Figure 2). To confirm our assay method, gel electrophoresis experiments have been carried out. As is shown in Figure 3A, new bands appear in lane 1 when both Dam MTase and Dpn I are added in the test solution, suggesting that a methylation reaction has taken place and the methylated DNA are then cut into small pieces. As a comparison, it can be observed that there is only one band of the original probe in lanes 2 and 3 when the Dam MTases are absent, indicating that no methylation/cleavage reaction occurs. Figure 3B shows the real-time UV-vis scan curves of two samples: curve 1 for AuNP/S1-2, Dpn I with the addition of Dam MTase, and curve 2 for AuNP/S1-2 with Dpn I. It can be observed that there is no signal enhancement in curve 2, indicating that Dpn I endonuclease will not cleave the DNA tethered on AuNPs in the absence of Dam MTase. Nevertheless, as is shown in curve 1, for the case that both Dpn I and Dam MTase are present, a rapid increase of A620/A520 can be detected. These experimental data also clearly confirm that AuNP aggregation as a result of the cleavage reaction cannot be initiated if the DNA methylation 232

Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

Figure 2. Changes of UV-vis spectra of AuNP/S1-2 within 8 min after the addition of Dam MTase (8.0 units/mL). Inset shows the visible color changes of the AuNPs.

Figure 3. (A) Gel electrophoresis images: (1) DNA, Dam MTase, and Dpn I; (2) DNA and Dpn I; (3) DNA alone. (B) Time scan spectra of AuNP/S1-2 with and without Dam MTase. Curves 1 and 2 are separately the cases that 10 units/mL Dam MTase is involved or not.

has not been achieved, which are in accordance with the results provided by the gel electrophoresis assay. Certainly, compared with electrophoresis, our method is much faster, which takes just about 6 min to finish. The initial rate of the methylation reaction can be obtained by determining the linear regression of the initial parts of the UV-vis scan curves.44 It is found that the rate of DNA methylation is directly related to the amount of Dam MTase used in the assay. Meanwhile, the detection range of Dam MTase in the current assay has been shown to be from 1.0 to 10 units/mL when the activity of Dpn I endonuclease is equal to 20 units (Figure 4). Under the reaction conditions mentioned above, the detection limit is 0.3 units/mL, which is much lower than the previous reports. Assay of the Inhibition of DNA MTase Activity. Since DNA methylation is closely related to our health and disease and inhibitors of DNA methylase have potential application in cancer therapy and antimicrobial infection, screening of inhibitors of DNA MTase receives more and more interest, and we have, thus, checked whether our simple and sensitive assay method can be employed for high-throughput screen of Dam MTase inhibitors. The mechanism is that, if transferring of the methyl group to the DNA residue is blocked in the presence of inhibitor, the specific site of the DNA molecules will not be recognized and cleaved; thus, the AuNPs will keep the state of dispersion. Experimental (44) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principle of Biochemistry; Worth Publishers: New York, 1993; pp 51-120.

Figure 4. Assay of the activity of Dam MTase. The curves from bottom to the top were obtained with different activities: 1.0, 2.0, 4.0, 6.0, and 10 units/mL. Inset shows the linear relationship between the initial methylation velocity rate and the activity of Dam MTase.

Figure 5. Inhibition of different concentrations of 5-fluorouracil on the activity of Dam MTase. Curves 1 and 2 are the time scan spectrum with and without 5-fluorouracil in the sample. Inset shows the inhibition efficiency of Dam MTase by 5-fluorouracil at different concentrations (0, 0.1, 0.2, 0.5, and 1.0 µM).

results show that the inhibitory effect of the inhibitors can be indeed quantitatively analyzed using the ratios of absorbance A620/A520 (Figure 5). Several reported Dam MTase inhibitors (benzyl penicillin, 5-fluorouracil, and mitomycin) have been used in our study.5,15 With the increase of the inhibitor concentration, the velocity of methylation will be decreased and the ratio of absorbance A620/A520 is observed

to increase more slowly. Experimental results also show that these inhibitors have a different effect on Dam methylase and 5-fluorouracil is the most effective inhibitor, which may decrease the activity of Dam MTase by about 55% (Figure S1 in the Supporting Information). The results are in good agreement with the previous reports.5,15 We have also checked the effect of the inhibitor concentration on the activity of methylase. As is shown in Figure 5, the inhibition effect of 5-fluorouracil on MTase is observed to enhance linearly with the concentration of the inhibitor. On the other hand, since there are two enzymes involved in our assay system, it is necessary to examine the effect of these inhibitors on another enzyme, Dpn I, besides MTase. The control experimental results have indicated that 5-fluorouracil does have inhibition effect on the activity of Dpn I endonuclease. However, when the concentration of the inhibitor is less than 1 µM, no influence can be observed. CONCLUSION In summary, we have developed a sensitive and simple method for a colorimetric assay of methylase activity using DNA functionalized AuNPs coupled with methylase and endonuclease. This new assay method takes advantage of specific recognition of the MTase and methyl-sensitive endonuclease as well as the unique optical properties of AuNPs. Compared with the traditional method to detect methylation, this proposed assay method can be very easily operated and less costly. Furthermore, since the access of enzymes to the DNA molecules modified on the well-dispersed AuNPs is much easier than to those embedded inside the aggregates of the AuNPs, compared with the approaches proposed in the recent studies, this strategy is more simple, and a lower detection limit can be obtained. In addition, this simple and sensitive assay method can be applied to screen inhibitors of Dam MTase, which might be a help for the discovery of antimicrobial drugs. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant No. 20575028) and the Natural Science Foundation of Jiangsu Province (Grant No. BK2008268). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 10, 2009. Accepted November 17, 2009. AC902198V

Analytical Chemistry, Vol. 82, No. 1, January 1, 2010

233