Sensitive Detection of DNA Methyltransferase Using Hairpin Probe

May 21, 2013 - Zhi-Mei Li , Zhao-Hua Zhong , Ru-Ping Liang , Jian-Ding Qiu ... cell cultures by monitoring the formation of 5-methylcytosine using HPL...
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Sensitive Detection of DNA Methyltransferase Using Hairpin ProbeBased Primer Generation Rolling Circle Amplification-Induced Chemiluminescence Ya-ping Zeng,† Juan Hu,† Yi Long,† and Chun-yang Zhang* Single-Molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong 518055, China ABSTRACT: DNA methyltransferases (MTases) catalyze the transfer of a methyl group from S-adenosylmethionine (SAM) to the 5-positon of cytosine in CpG islands, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Despite the development of various methods for the MTase assay, most of them are laborious and costly with poor sensitivity. Herein, we develop a highly sensitive chemiluminescence method for the MTase assay using hairpin probe-based primer generation rolling circle amplification (PG-RCA). In the presence of DNA adenine methylation (Dam) MTase, the methylation-responsive sequence of hairpin probe is methylated and cleaved by the methylation-sensitive restriction endonuclease Dpn I. The cleaved hairpin probe then functions as a signal primer to initiate PG-RCA reaction by hybridizing with the circular DNA template, producing a large number of horseradish peroxidasemimicking DNAzyme chains, which can catalyze the oxidation of luminal by H2O2 in the presence of hemin to yield distinct chemiluminescence signal. While in the absence of Dam MTase, neither methylation/cleavage nor PG-RCA reaction can be initiated and no chemiluminescence signal is observed. The proposed method exhibits a wide dynamic range from 0.025 to 400 U/mL and an extremely low detection limit of 1.29 × 10−4 U/mL, which is superior to most conventional approaches for the MTase assay. This method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

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DNA MTase activity, such as the hairpin DNA probe-based fluorescence method21,22 and the gold nanoparticle-based enzyme-linkage reaction assay.23,24 However, the gold nanoparticle-based enzyme-linkage reaction assay involves the complicated nanoparticle preparation and functionalization,23 and the hairpin DNA probe-based fluorescence method requires expensive double-labeled DNA probes which are difficult to synthesize.21 Recently, isothermal amplification techniques, such as rolling circle amplification (RCA),25 strand displacement amplification (SDA),26 and nicking enzyme signal amplification (NESA),27 are frequently used for an enzyme assay due to their wide variety of choices and significant signal amplification. Among these amplification methods, the SDA28 and NESA29 methods have been used for the DNA MTase assay. The SDA-based colorimetric method allows simple visualization of MTase activity, but the poor detection limit of the colorimetric method and the linear amplification characteristic of SDA adversely affect the detection sensitivity.30 The NESA-based method exhibits improved sensitivity, but it requires the initial heating to denature the molecule beacon and thus is not truly isothermal.29 Consequently, the development of highly sensitive, specific, and low-cost methods for the DNA MTase assay still remains a great challenge.

NA methylation is an epigenetic modification in human genomes and plays an important role in the regulation of gene expression.1 Recent research demonstrates that DNA methylation might be used as a potential biomarker for early clinical diagnosis.2−4 DNA methylation occurs at the C-5/N-4 positions of cytosine and at the N-6 position of adenine and is catalyzed by DNA methyltransferases (MTases).5−7 During this aberrant methylation process, the DNA MTase acts as a crucial participator to transfer a methyl group from S-adenosylmethionine (SAM) to the C5-position of cytosine in CpG islands.8 Although a variety of methods have been developed for genome-wide methylation extent analysis, gene-specific methylation analysis, and screening for new methylated sites,9−13 the relevant studies that focus on the DNA MTase are rare. So far, four human DNA MTases have been discovered to preferentially methylate different kinds of DNA and play different roles in keeping DNA methylation patterns.14,15 Notably, the DNA MTases have been treated as a potential target for anticancer drugs.16,17 Therefore, the development of a sensitive method for the MTase assay is of significance for both fundamental biochemical research and drug discovery. The conventional methods for the DNA MTase assay include high-performance liquid chromatography (HPLC),18 gel electrophoresis,19 and radioactive labeling.20 Although these methods are well established, most of them are laborious and time-consuming with the involvement of radioactive materials. To overcome these disadvantages, some new approaches have been developed for real-time and nonradioactive detection of © XXXX American Chemical Society

Received: April 17, 2013 Accepted: May 21, 2013

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Methylation and Cleavage of Hairpin Probe. The methylation of hairpin probe was carried out in 50 μL of reaction mixtures containing various amounts of Dam MTase, 2 μM hairpin probe, 1× Dam buffer (50 mM Tris−HCl (pH 7.5), 10 mM NaCl, 10 mM EDTA, 5 mM 2-mercaptoethanol), and 160 μM SAM. After methylation, 50 μL of methylation products was added to 150 μL of buffer solution containing 1× NEB buffer 4 (50 mM KAc, 20 mM Tris−Ac, 10 mM Mg(Ac)2, 1 mM DTT (pH 7.9)) and 10 U Dpn I for the cleavage reaction. The mixture was incubated at 37 °C for 1.5 h, followed by inactivation at 80 °C for 20 min. Preparation of Circular Probes. Circular probes were prepared by ligation reaction in 20 μL of buffer containing 1× T4 ligase buffer (6.6 mM MgCl2, 10 mM DTT, 0.1 mM ATP, 66 mM Tris−HCl (pH 7.6)), 1 μM primer, 1 μM linear padlock probe, and 50 U T4 ligase at 16 °C overnight after initial denaturation at 95 °C for 5 min. After ligation, 10 μL of products was added to 10 μL of exonuclease mixture containing 1 mM DTT, 6.7 mM MgCl2, 67 mM glycine−KOH (pH 9.5), 10 U Exonuclease I, and 20 U Exonuclease III, and the mixture was incubated at 37 °C for 2 h, followed by inactivation at 95 °C for 10 min. The digestions were purified by Dr.GenTLE Precipitation Carrier (TaKaRa Biotechnology Co., Ltd., Dalian, China). PG-RCA Reaction and Chemiluminescence Detection. PG-RCA reaction was performed at 60 °C for 90 min in 20 μL of reaction solution containing 20 mM Tris−HCl buffer (pH 8.8), 10 mM (NH4)2SO4, 10 mM KCl, 6 mM MgSO4, 400 μM dNTP, 0.1% Triton X-100, 30 nM circular probe, 0.16 U Vent (exo-) DNA polymerase, 3 U Nb.Bam I, and 1 μL of digestion products. The luminol solution (0.5 mM) and hemin solution (75 nM) were prepared by adding luminol and hemin to the mixture of 15 μL of PG-RCA products, 15 μL of ddH2O, and 75 μL of 2× HEPES buffer (40 mM HEPES, 600 mM NaCl (pH 8.0)), respectively. The mixture was then incubated at room temperature for 30 min to allow the DNA to fold into the enzymatically active quadruplex structures of DNAzyme. After the addition of 30 μL of H2O2 (10 mM) to the mixture, the chemiluminescence signal was recorded on a GloMax 96 Microplate Luminometer (Promega, Madison, WI, USA) with a time interval of 1.5 s. Inhibition Assay. The mixture of 1× Dam buffer (50 mM Tris−HCl (pH 7.5), 10 mM NaCl, 10 mM EDTA, 5 mM 2mercaptoethanol), 1 μM hairpin probe substrate, and different concentrations of 5-fluorouracil was preincubated at 37 °C for 15 min. Then, 8 U Dam MTase and 160 μM SAM were added into the solution, followed by incubation at 37 °C for 2 h. The chemiluminescence intensities of PG-RCA products were recorded on a GloMax 96 Microplate Luminometer (Promega, Madison, WI, USA) for the calculation of relative activity of Dam MTase in the presence of inhibitors. Real-Time Fluorescence Detection and Gel Electrophoresis. The real-time fluorescence measurements of PGRCA reaction were performed in a Roche Light Cycler Nano (Switzerland) with 1× SYBR Green I as the fluorescent indicator, and the fluorescence intensity was monitored at a time interval of 40 s. A 10% nondenaturating polyacrylamide gel electrophoresis (PAGE) was carried out in 1× TBE (9 mM Tris−HCl (pH 7.9), 9 mM boric acid, 0.2 mM EDTA) with 1× SYBR Green I as the fluorescent indicator. A 1% agarose gel electrophoresis analysis of PG-RCA products was carried out in 1× TAE (40 mM Tris−ethylic acid, 2 mM EDTA) with 1× SYBR Green I as the fluorescent indicator. The images were

Herein, we develop a highly sensitive method for the DNA MTase assay using hairpin probe-based primer generation rolling circle amplification (PG-RCA)-induced chemiluminescence. Recently, a variety of rolling circle amplification (RCA) methods have been introduced,31−35 including the linear rolling circle amplification (LRCA)31,32 and the hyper-branched RCA.33,34 The LRCA is a linear amplification method and suffers from relatively low amplification efficiency.36 The hyperbranched RCA is an exponential amplification method, but it needs two primers and might suffer from the nonspecificity.13 In contrast, PG-RCA can convert the conventional LRCA to an exponential amplification with the simple design of circular DNA template.37,38 In this study, we employ for the first time the hairpin probe-induced PG-RCA reaction for the DNAzyme generation and DNAzyme-driven chemiluminescence for signal readout. The DNAzyme can form a special G-quadruplex in the alkaline condition,39 which can act as a biocatalytic label for the chemiluminescence signal enhancement with the insertion of hemin.40 Taking advantage of the exponential amplification efficiency of PG-RCA and the intrinsically high sensitivity of DNAzyme-driven chemiluminescence, the proposed method provides an ultrasensitive platform for the MTase assay with a wide dynamic range from 0.025 to 400 U/mL and an extremely low detection limit of 1.29 × 10−4 U/mL and can be further applied for the screening of antimicrobial drugs.

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EXPERIMENTAL SECTION Materials. All oligonucleotides were HPLC-Purified and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The sequences of oligonucleotides are listed in Table 1. Table 1. Sequences of Hairpin Probe, Linear Padlock Probe, Primer, and Synthesized DNAzymea note

sequence (5′-3′)

hairpin probe

ACT TAT CAG CTT AAG GAT CTT CTC GAC AGC TTG CTG AAG ATC CTT AAG CTG ATA AGT P-CTG TCG AGA AGA ATC GAA TGC AAA AAC CCA ACC CGC CCT ACC CAA ATC TTC AGC AAG TCT TCT CGA CAG CTT GCT GAA GA GGG TAG GGC GGG TTG GG

linear padlock probe primer synthesized DNAzyme a

In the hairpin probe, the binding region of circular probe is shown in italic. In the linear padlock probe, the binding region of primer is shown in boldface, and the nicking site is shown in the underline. The P in the linear padlock probe indicates a 5′ phosphate modification.

Methyltransferase of DNA adenine methylation (Dam), methyltransferases of DNA cytosine methylation (M.sssI), endonuclease of Dpn I, S-adenosylmethionine (SAM), Vent (exo-) polymerase, Nb.BsmI, and the corresponding buffer solutions were purchased from New England Biolabs (Beverly, MA, USA). T4 DNA ligase, Exonuclease I, and Exonuclease III were purchased from TaKaRa Biotechnology, Co. Ltd. (Dalian, China). Hemin, luminal, and 4-(2-hydroxyethyl) piperazine-1ethanesulfonic acid sodium salt (HEPES) were obtained from Sigma-Aldrich Co (St. Louis, MO). Luminol solution (25 mM) was prepared by the use of 0.1 mM NaOH as the solvent. A stock solution of hemin (25 μM) was prepared with dimethysulfoxide (DMSO) and stored at −20 °C in the dark. Other chemicals were of analytical grade and were used without further purification. Ultrapure water obtained from a Millipore filtration system was used throughout. B

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Scheme 1. Schematic Illustration of Dam MTase Assay Using Hairpin Probe-Based Primer Generation Rolling Circle Amplification (PG-RCA)-Induced Chemiluminescence

reaction of LRCA in the presence of Vent (exo-) polymerase, producing the long repeat sequence of circular probe. This amplified repeat sequence is nicked by the addition of Nb.BsmI and produces many new primers for the circular template. Each new primer can initiate a new extension circle and produce large numbers of DNAzyme in a way of exponential amplification. In the third step, the DNAzyme folds into Gquadruplex structure in the alkaline condition and binds with the hemin molecule, yielding a biocatalytic G-quadruplex/ hemin structure and generating a chemiluminescence signal in the presence of H2O2 and luminol.40 Consequently, by the introduction of an exponential PG-RCA reaction and ultrasensitive DNAzyme chemiluminescence readout, the limitation of low efficiency of Dam MTase and Dpn I reactions42 can be efficiently overcome, and the proposed method can be used for sensitively monitoring the DNA methylation catalyzed by MTase. To demonstrate the necessity for the coexistence of Dam MTase and Dpn I, a nondenaturating PAGE experiment with SYBR Green I as the fluorescent indicator was performed. As shown in Figure 1A, there is only one band of the original probe when either Dpn I or Dam MTase is absent (Figure 1A, lanes 1 and 2), indicating that no methylation/cleavage reaction occurs. When both Dam MTase and Dpn I are present, a new band of 17-bp base appears (Figure 1A, lane 3), suggesting the occurrence of methylation and cleavage reaction.21 To investigate the necessity of nicking enzyme for the PGRCA, a real-time fluorescence measurement of PG-RCA reaction was performed with the involvement of three enzymes of Dam MTase, Dpn I, and nicking enzyme. As shown in Figure 1B, in the absence of either Dam MTase or Dpn I, the hairpin probe cannot be cleaved and no signal primer is produced even though it is in the presence of nicking enzyme. As a result, no

acquired with a Kodak Image Station 4000MM (Woodbridge, CT).

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RESULTS AND DISCUSSION Principle of Methyltransferase Assay. The proposed MTase assay is based on the specificity of some restriction endonucleases for methylated sites and involves an efficient dual-enzyme (Dam MTase and Dpn I endonuclease) coupling reaction.41 To keep it stable under the PG-RCA reaction condition, a hairpin probe (Table 1) with an annealing temperature of more than 60 °C was designed. The circular probe is generated by the ligation of linear padlock probe and primer (Table 1), and it contains three sections: (1) a binding site for signal primer, (2) a nicking site of Nb.BsmI, and (3) a complementary sequence of horseradish peroxidase-mimicking DNAzyme. As shown in Scheme 1, the Dam MTase assay involves three principal processes: (1) a Dam MTase and Dpn I endonuclease coupling reaction, (2) a PG-RCA reaction at 60 °C, and (3) chemiluminescence detection. In the first step, a hairpin probe with the palindromic sequence of 5′-G-A-T-C-3′ in the stem part which functions as the reaction substrate is catalyzed to a methylated hairpin probe (5′-G-mA-T-C-3′) by Dam MTase in the presence of SAM.7 Then, this methylated hairpin probe is cleaved into two parts by the methylation-sensitive restriction endonuclease Dpn I:41 one is a 23-base hairpin DNA containing a loop (Table 1, in italic) and a 5-base blunt terminus, and the other one is a 17-bp double stranded DNA (dsDNA). In the second step, the cleaved 23-base hairpin DNA is changed into single-stranded DNA (ssDNA) because its Tm value of 28 °C is lower than the PG-RCA reaction temperature of 60 °C. Then, the 23-base ssDNA functions as a signal primer to bind with the circular template and initiates a cascade C

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Figure 1. (A) Nondenaturating PAGE analysis of the digestion products of Dam and Dpn I. The products are separated by 10% PAGE and stained by SYBR green I. Lane M is the DNA ladder marker; Lane 1 represents the products in the presence of hairpin probe and Dam MTase; Lane 2 represents the products in the presence of hairpin probe and Dpn I; Lane 3 represents the products in the presence of hairpin probe, Dam MTase, and Dpn I. (B) Real-time fluorescence monitoring of PG-RCA reaction with SYBR Green I as the fluorescent indicator in the presence of Dam and nicking enzyme (blue line), Dpn I and nicking enzyme (purple line), Dam and Dpn I (green line), and Dam and Dpn I and nicking enzyme (red line), respectively. (C) Electrophoretic identification of amplification products of the PG-RCA reaction in the presence of Dam and nicking enzyme (a), Dpn I and nicking enzyme (b), Dam and Dpn I and nicking enzyme (c), Dam and Dpn I (d), respectively. (D) Chemiluminiscence analysis of amplification products of PG-RCA reaction in the presence of Dam and nicking enzyme (a), Dpn I and nicking enzyme (b), Dam and Dpn I and nicking enzyme (c), and Dam and Dpn I (d), respectively. The concentration of Dam MTase used in all experiments is 40 U/mL. Error bars show the standard deviation of three experiments.

PG-RCA amplification occurs and no observable fluorescence signal is observed (Figure 1B, blue and purple lines). In contrast, in the presence of Dam MTase, Dpn I, and nicking enzyme, the fluorescence intensity increases with the reaction time in a sigmoidal fashion (Figure 1B, red line), indicating that the PG-RCA reaction proceeds in a way of exponential amplification.43,44 Notably, in the presence of Dam MTase and Dpn I but without nicking enzyme, the polymerization reaction proceeds in a linear way, resulting in a relatively low fluorescence intensity (Figure 1B, green line). The amplification products are further analyzed by 1% agarose gel electrophoresis (Figure 1C). The distinct bands appear only in the presence of Dam MTase, Dpn I, and nicking enzyme (Figure1C, line c), but no distinct band appears in the control groups in the absence of either Dpn I (Figure 1C, line a) or Dam MTase (Figure 1C, line b). Notably, in comparison with that of PG-RCA, the amplification efficiency of LRCA is so low that no distinct band is observed (Figure 1C, line d).37 The above results are further confirmed by the chemiluminiscence analysis of amplification products of PG-RCA (Figure 1D). In

the presence of nicking enzyme but without either Dpn I or Dam MTase, no significant chemiluminiscence (CL) signal is observed (Figure 1D, a and b). However, in the presence of nicking enzyme, Dam MTase, and Dpn I, a high chemiluminiscence signal with 31-fold increase is observed (Figure 1D, c). Notably, the chemiluminiscence signal decreases by as much as 7-fold in the presence of Dam MTase and Dpn I but without nicking enzyme (Figure 1D, d) as compared with that in the presence of Dam MTase, Dpn I, and nicking enzyme (Figure 1D, c). These results clearly demonstrate that the introduction of nicking enzyme can convert a linear rolling circle amplification to an exponential amplification,37 significantly improving the amplification efficiency of the PG-RCA reaction. Notably, in contrast to the LRCA reaction which produces long concatencated sequences, the PG-RCA produces small fragments which benefit the complete folding of DNAzymes into G-quadruplexes. Optimization of Experimental Condition. The chemiluminescence signal generated by H2O2 and luminal is dependent on the amount of the hemin molecule bound to D

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Figure 2. (A) Variance of chemiluminescence value ratio of I/I0 with the concentration of hemin. I and I0 are the chemiluminescence signals with and without synthesized DNAzyme, respectively. (B) Variance of chemiluminescence value ratio of I/I0 with the luminol concentration. I and I0 are the chemiluminescence signals with and without synthesized DNAzyme, respectively. (C) Variance of the chemiluminescence intensity with the amounts of vent (exo-) polymerase. (D) Variance of the chemiluminescence intensity with the reaction time of PG-RCA. Error bars show the standard deviation of three experiments.

the cooperation of vent (exo-) polymerase and Nb.BsmI nickase.37 Previous research demonstrated that the concentration of Nb.BsmI had no influence upon the amplification efficiency;37 thus, we simply investigated the influence of vent (exo-) polymerase upon the chemiluminescence signal with a fixed amount of Nb.BsmI nickase (3 U). As shown in Figure 2C, the chemiluminescence signal increases with the increasing amount of vent (exo-) polymerase from 0.04 to 0.16 U, and the highest chemiluminescence signal was obtained at 0.16 U vent (exo-) polymerase. This result is consistent with that of previous research.26 Therefore, 0.16 U vent (exo-) polymerase is used in the subsequent research. We further investigated the influence of PR-RCA reaction time upon the chemiluminescence signal. As shown in Figure 2D, the chemiluminescence signal increases rapidly with the PG-RCA reaction time from 15 to 90 min and reaches a plateau beyond 90 min, which might result from the termination of PGRCA reaction due to either the exhaustion of RCA substrates or the inactivation of the polymerase enzyme. Therefore, the PGRCA reaction time of 90 min is used in the subsequent research. Sensitivity and Selectivity of the Proposed Method. To investigate the analytical performance of the proposed method, we measured the Dam MTase at various concentrations under the optimal conditions. Figure 3A shows the variance of chemiluminescence intensity with the concentration of Dam MTase. As the concentration of Dam MTase increases, large numbers of primers are produced for PG-RCA to synthesize the DNAzyme, resulting in the increase of

the products of PR-RCA and is significantly influenced by the amount of polymerase,37 reaction time of PG-RCA,38 and hemin and luminol concentrations.45 To achieve a good performance, the experimental condition of the proposed method was carefully optimized (Figure 2). To investigate the influence of hemin concentration upon the chemiluminescence signal, a synthesized DNAzyme (Table 1) with various concentrations was used to imitate the products of PG-RCA reaction.46 Due to the dependence of catalytic kinetic of luminol−H2O2 reaction upon hemin concentration,39 the chemiluminescence signal increases with the increasing hemin concentration in both the test group with the presence of synthesized DNAzyme (I) and the control group without the presence of synthesized DNAzyme (I0). In order to optimize the hemin concentration, the variance of chemiluminescence ratio value of I/I0 with the hemin concentration was evaluated. As shown in Figure 2A, the value of I/I0 increases with the increase of hemin concentration from 0.75 to 75 nM, followed by the decrease beyond the hemin concentration of 75 nM. Therefore, 75 nM hemin is used in the subsequent research. The luminol concentration is further optimized, too. As shown in Figure 2B, the value of I/I0 increases with the increase of luminol concentration from 0.05 to 0.5 mM, followed by the decrease beyond the luminol concentration of 0.5 mM. Thus, 0.5 mM luminol is used in the subsequent research. In theory, a high-efficiency enzyme and a long PR-RCA reaction time are expected to generate large numbers of complementary segments of circular template for enhanced signal amplification. The efficiency of PG-RCA relies mainly on E

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responsive DNAzyme colorimetric assay24 and the real-time fluorescence method.21 Such extremely high sensitivity of the proposed method can be attributed to following two factors: (1) the high amplification efficiency of PG-RCA,37 which has improved by 7 orders of magnitude as compared with that of HRCA13 and 8 orders of magnitude as compared with that of SDA;26 (2) the high sensitivity and wide dynamic range of the chemiluminescence method whose detection limit can reach zeptomole47 and sensitivity has improved by as much as 4 orders of magnitude as compared with that of colorimetric method.47 In addition, the proposed method exhibits a good reproducibility with a coefficient of variance (CV) of 3.4% for 5 parallel experiments. To investigate the selectivity of the proposed method, M.SssI MTase was introduced as an interference enzyme. M.SssI MTase can methylate all cytosine residues within the doublestranded dinucleotide recognition sequence of 5′-C-G-3′.48 Due to the specific site recognition of Dam MTase toward its substrate,41 the proposed method can easily discriminate Dam MTase from M.SssI MTase. As shown in Figure 3C, significant chemluminescence enhancement is observed in the presence of Dam MTase. In contrast, no distinct chemluminescence signal is observed in the presence of M.SssI MTase, suggesting the high selectivity of the proposed method toward Dam MTase. Inhibition Assay. Since Dam MTase plays an important role in the virulence of bacterial pathogens and Dam MTase inhibitors are likely to have broad antimicrobial action, Dam MTase has become a promising target for antimicrobial drug development.16,17 In this research, we choose 5-fluorouracil as the model inhibitor to investigate the feasibility of proposed method for the screening of antimicrobial drugs. 23,24 Considering two facts of (1) the cooperation of Dam MTase methylation and Dpn I endonuclease-induced signal primers for PG-RCA and (2) the aim of investigating the effect of inhibitors upon the Dam MTase activity, we should first exclude the effect of inhibitors upon Dpn I. Previous research demonstrated that 5-fluorouracil had no inhibition effect upon the Dpn I activity when the concentration of 5-fluorouracil was below 10 μM;24 thus, 5-fluorouracil with concentration less than 10 μM was used in this research. The chemiluminescence signal in response to different concentrations of 5-fluorouracil was simultaneously obtained by the GloMax 96 Microplate Luminometer, and the relative activity (RA) of Dam MTase can be quantitatively calculated using the following equation:49

Figure 3. (A) Chemiluminescence signal obtained from the PG-RCA triggered by different concentrations of Dam MTase. (B) Linear relationship between the chemiluminescence intensity and the logarithm of Dam MTase concentration. (C) Selectivity of the proposed method. The concentration of Dam MTase is 50 U/mL, and the concentration of M.SssI MTase is 50 U/mL. Error bars show the standard deviation of three experiments.

RA =

chemiluminescence intensity correspondingly. An exponential curve is obtained between the chemluminescence intensity and concentration of Dam MTase from 0.025 to 400 U/mL (Figure 3A). Notably, in logarithmic scales, the chemiluminescence intensity exhibits a linear correlation with the concentration of Dam MTase over a range of 2 orders of magnitude from 0.025 to 2.5 U/mL (Figure 3B). The regression equation is Ic = 147614.4 + 34572.3log10C with a correlation coefficient of 0.997, where Ic and C represent the chemiluminescence intensity and the concentration of Dam MTase (U/mL), respectively. The detection limit is calculated to be 1.29 × 10−4 U/mL by evaluating the average response of blank plus three times the standard deviation. Notably, the sensitivity of the proposed method has improved by as much as 3 orders of magnitude as compared with those of the methylation-

I i − I0 × 100% I t − I0

where I0, It, and Ii are the chemiluminescence intensity in the absence of Dam MTase, in the presence of Dam MTase, and in the presence of both Dam MTase and 5-fluorouracil, respectively. As shown in Figure 4, the obtained relative activity of Dam MTase decreases with the increase of 5fluorouracil concentration. The IC50 value is the inhibitor concentration required to reduce enzyme activity by 50%. On the basis of the plot of relative activity of Dam MTase versus 5fluorouracil concentration (Figure 4), the IC50 of 5-fluorouracil is calculated to be 1.42 ± 0.07 μM. Therefore, the proposed method has the potential to be applied for the screening of antimicrobial drugs. F

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Figure 4. Variance of relative activity of Dam MTase with 5fluorouracil concentration. Error bars show the standard deviation of three experiments.

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CONCLUSIONS In summary, we have developed a simple, highly sensitive chemiluminescence method for the detection of MTase activity using hairpin probe-based primer generation rolling circle amplification. In comparison with the fluorescent-labeled hairpin probes for the MTase assay, the hairpin probe used in the proposed method does not require the expensive and labor-intensive fluorescent modification, and the whole reaction can be performed in an isothermal condition without the requirement for high-precision thermal cycling, making the proposed method more simple and cost-effective. Notably, taking advantage of exponential amplification efficiency of PGRCA and the intrinsically high sensitivity of DNAzyme-driven chemiluminescence, the proposed method exhibits excellent sensitivity with a detection limit of 1.29 × 10−4 U/mL, which has improved by as much as 3 orders of magnitude as compared with those of conventional approaches for the MTase assay.21,24 Importantly, the proposed method can be used for the screening of antimicrobial drugs and might be further applied for early clinical diagnosis.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 2010CB732600), the National Natural Science Foundation of China (Grant Nos. 21075129 and 21205128), the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development (Grant No. (2012) 433), and Award for the Hundred Talent Program of the Chinese Academy of Science.

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