Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy

Analytica Chimica Acta xxx (2016) 1e7

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Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy Linqun Zhang a, b, 1, Yuanjian Liu a, 1, Ying Li a, Yuewu Zhao a, Wei Wei a, *, Songqin Liu a, ** a

Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, PR China b Center for Analysis and Testing, Nanjing Normal University, Nanjing, 210023, PR China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


An electrochemical strategy for sensing DNA MTase activity was proposed.
Signal amplification was achieved by mimic-hybridization chain reaction.
High specificity was obtained based on synergistic effect of Hap II and EXO III.
The strategy has great potential be applied in methylation-based disease diagnosis.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 April 2016 Received in revised form 16 May 2016 Accepted 23 May 2016 Available online xxx

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwichtype assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6]3þ, RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was nonmethylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL1 to 10 U mL1, with a detection limit down to 0.03 U mL1. Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. © 2016 Published by Elsevier B.V.

Keywords: DNA methyltransferase activity Mimic-hybridization chain reaction Signal amplification Differential pulse voltammetry

1. Introduction

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (W. Wei), [email protected] (S. Liu). 1 L.Q. Zhang and Y.J. Liu have contributed equally to this work.

DNA methylation in the mammalian genome occurs after DNA synthesis by enzymatic transfer of a methyl group from the methyl donor, S-adenosylmethionine (SAM), to the 5 position of cytosine residues (Fig. S1). Currently, DNA methylation is believed to involve in regulating many cellular processes, including X-chromosome inactivation, genomic imprinting, chromosome stability, and gene

http://dx.doi.org/10.1016/j.aca.2016.05.044 0003-2670/© 2016 Published by Elsevier B.V.

Please cite this article in press as: L. Zhang, et al., Sensitive electrochemical assaying of DNA methyltransferase activity based on mimichybridization chain reaction amplified strategy, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.05.044

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transcription [1]. Consequently, it is correlated with a larger majority of diseases, such as various cancers [2e5], aging [6,7], fibrosis [8], obesity [9], and neuropsychiatric disorder [10]. Accordingly, the activity of MTase may influence DNA methylation level which has great effects on people health [11e13]. Thus, a sensitive and specific method for MTase activity detecting and inhibition screening is very significant to diagnose and treat diseases early, as well as to predict the performance of the drug. So far, some methods have been developed for the detection of MTase activity. One of the most frequently used approaches is bisulfite conversion which can reproducibly change unmethylated cytosine to uracil but leaves methylated cytosine unchanged. However the procedure of it is relatively complicated and poorly suited for routine diagnostics [14,15]. The restriction enzyme assay is also an amazing method due to its powerful selectivity, in which the restriction enzymes can sensitively recognize the methylation position and catalyze the cleavage of the specific base sequence. When the cytosine in the specific sequence is methylated, the cleavage reaction is inhibited. Based on the restriction enzyme assay, many novel methods are developed recently for MTase detection, including colorimetric assay [16e18], fluorescence [19e22] and luminescence-based approach [23,24], circular dichroism [25], electrochemistry [26e31] and so on [32,33]. Among them, electrochemistry was the most attractive method for MTase activity detection owing to its remarkable features of high sensitivity, inexpensive instrument, low cost and portability [34]. Ai recently employed a new type of DNA functionalized nano/mesoporous silica to detect DNA MTase activity with G-quadruplex as a lock for electrochemical signal molecule releasing [31]. Our group designed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on the deposition of polyaniline catalyzed by HRP-mimicking DNAzyme [30]. In order to enhance the assay sensitivity, some nanostructured materials, such as graphene oxide (GO), metal-NPs and so on, have been coupled into various strategies to amplify the electrochemical signal for detection of DNA methylation and MTase activity. Qiu and co-workers employed signal amplification of sliver nanoparticles, GO and luminol composites to enhance electrochemiluminescence signal [24] and got satisfied results. Au-NPs were also frequently used as signal amplified element to improve the assay sensitivity [27,32,35]. Hybridization chain reaction (HCR) is an enzyme-free, room temperature linear amplification approach which is simple in operation and cost-effective only by initiators triggering alternating hybridization of two hairpin DNA. It has been re-engineered to improve the amplification efficiency by many scientists [36,37]. Zhu [38] reported a mimic-HCR that can produce dual signal amplification for spherical nucleic acids gold nanoparticles act as both initiators of HCR and primary amplification element. Herein, we combined “sandwich-type” assembly [35] with mimic-HCR strategy to develop a simple, rapid, and highly sensitive assay for detecting the activity of M.SssI MTase and inhibitor by using RuHex as indicator, endonuclease HpaII and exonuclease III as assisted enzyme. DNA-AuNPs was modified on the Au electrode by“sandwich-type ” assembly. Without the methylation of M.SssI MTase, the special sequence of 50 -CCGG-30 in sandwiched hybridization could be recognized, cleaved by HapII, and further digested by ExoIII for it is a double-strand-specific exodeoxyribo-nuclease which can catalyze the stepwise removal of mononucleotides from 30 to 50 [39,40]. As a result, DNA-AuNPs couldn't attach to the Auelectrode surface and no mimic-HCR occurred so that the differential pulse voltammetry (DPV) of RuHex had no obvious current. However, when the cleavage was blocked after the methylation of M.SssI MTase, DNA-AuNPs on the Au-electrode surface could induce mimic-HCR, resulting in high DPV current. Thus, the difference of signals are related to MTase activity, which can be used to

construct an electrochemical sensor for detection of MTase activity. In addition, the mimic-HCR formed many long nicked dsDNA for RuHex inserting to achieve 4.5 times of signal amplification. Meanwhile, the transfer of electrons from RuHex to Au electrode could be accelerated by AuNPs. 2. Experimental 2.1. Materials and instrumentation Restriction endonucleases HpaII, exonuclease III, S-adenosylmethionine, CpG methyltransferase (M.SssI, MTase) were purchased from Thermo Scientific (USA). 6-Mercapto-1-hexanol (MCH), hexaammineruthenium (II) chloride ([Ru(NH3)6]3þ, RuHex), 5-azacytidine (5-Aza), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and bis(p-sulfonatophenyl) phenylphosphine dihydrate dipotassium salt (BSPP) were obtained from Sigma-Aldrich. Tris (hydroxymethyl) aminomethane (Tris), procaine, KCl, NaCl, and MgCl2$6H2O were of analytical grade and purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). All chemicals were used without further purification. The sequences of all the oligonucleotides synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China) were listed in Table 1. These oligonucleotides included capture DNA (C1), link DNA (L2), probe DNA (P3), and DNAs for mimic-HCR (H4, H5). A 30 mM Tris-HCl (pH 7.9) containing 100 mM NaCl, 10 mM MgCl2 was employed as hybridization and mimic-HCR buffer (Buffer I). A 10 mM PB (pH 7.4) containing 100 mM KCl was used as washing buffer (Buffer II). A 10 mM PB (pH 7.4) containing 100 mM KCl and 5 mM RuHex was used as the electrochemical buffer (Buffer 4þ III). A 0.1 M PBS (pH 7.4) containing 5 mM Fe (CN)3þ 6 /Fe (CN)6 and 0.1 M KCl was employed as the electrochemical impedance spectroscopy (EIS) buffer (Buffer IV). Milli-Q water (18 MU at 25  C, Barnstead, Thermo Scientific, USA) was used throughout the experiments. All oligonucleotides were used as provided and were dissolved in Buffer I to give the stock solutions of 10 mM. TEM (JEM-2010, Hitachi, Japan) was used to characterize the morphologies of monodispersed AuNPs. UVevis spectra were measured on a circular dichroism spectrometer (Chirascan, Applied Photophysics Ltd., England), cyclic voltammetry (CV) and differential pulse voltammetric (DPV) were performed with a model CHI 660C electro chemical work station (Shanghai Chenhua quipments, China). Electrochemical impedance spectroscopy (EIS) measurements were performed on a VersaStat 3 electrochemical workstation (Princeton Applied Research). The three-electrode system was consisted of a KCl saturated calomel electrode (SCE) as the reference, a platinum electrode as the counter electrode, and a gold electrode (2 mm diameter) as the working electrode. 2.2. Preparation of P3 modified AuNPs Firstly, 13 nm AuNPs were prepared according to our previous

Table 1 Sequences of the oligonucleotides used in this study.a Oligo name

Sequence (50 e30 )

C1 L2 P3 H4 H5

50 eSHe(CH2)6-TTTTTTCTGTGCGCCGGTCTCTCC-30 50 -ATCCGTCGAGCAGAGTTGGAGAGACCGGCGCACAG-30 50 -AACTCTGCTCGACGGATTTTTTTTe(CH2)6eSH-30 50 -AATCCGTCGAGCAGAGTTAACTCTGCTCGAGTAGTG-30 50 -AACTCTGCTCGATCTAGACACTACTCGAGCAGAGTT-30

a The bold font of CCGG was the recognized bases by M.SssI MTase and restriction endonuclease Hpa II.

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work [41] and their concentration was estimated to be ~10 nM. Secondly, 1 mg BSPP was added in 1 mL AuNPs solution to open sulfhydryl bond. This reaction was allowed to gently shake for over 12 h at room temperature. Then AuNPs were dissolved in 0.5 mL water again after being centrifuged for 20 min at 13, 200 rpm and 10  C. Thirdly, 20 mL of 100 mM P3 was added in AuNPs solution, then 50 mL of 1 M NaCl was added quickly to stabilize the colloid solution. The solution was allowed to stand for another 12 h so that P3 could coated onto the surface of AuNPs by SeAu interaction. Then it was purified by centrifuging (13, 200 rpm) for 15 min twice to remove unbounded P3. The P3 modified AuNPs (P3-AuNPs) was re-dispersed in 1 mL Buffer I and this conjugate was stored at 4  C for further use. 2.3. Preparation of C1 modified gold electrode The 2 mm diameter gold electrode (CH Instruments Inc., Shanghai, China) was treated with piranha solution (H2O2:H2SO4 ¼ 3:7 v/v) for 10 min. Then, electrode was rinsed with Buffer II and dried under nitrogen flow. The clean gold electrode was immersed in the mixture of 1 mM C1 in Buffer I and 500 mM TCEP for 12 h at room temperature to active the thiolated C1. Subsequently, the resulting electrode was rinsed thoroughly with Buffer II in order to remove the physically adsorbed DNA and incubated in 1 mM MCH for 1 h to eliminate nonspecific binding. The gold electrode modified by C1 (C1eAu) was now prepared for further use. 2.4. Analysis procedure 5 mL of L2 was dropped on to the C1 modified electrode surface for 1 h at 37  C, and L2 was assembled on the electrode by the hybridization of C1 and L2 (C1/L2). The methylation of C1/L2 was performed in Buffer I containing 150 mM SAM and M.SssI MTase of various concentrations (from 0 to 60 U mL1) at 37  C for 2 h. The cleavage and digestion reaction was performed at 37  C in Buffer II with 40 U mL1 HpaII and 50 U mL1 ExoIII for 1 h. After washed by Buffer II thoroughly, the electrode was incubated with another 5 mL of P3-AuNps for 1 h at 37  C. Then, 5 mL of 1 mM H4 and H5 were cast onto the surface of the above modified gold electrode for incubation 1 h at 37  C. After extra H4 and H5 were removed by Buffer II, the sensor was immersed in Buffer III to perform electrochemical measurements at room temperature. The DPV curves were background-subtracted using ORIGIN 8.5 (Microcal Software, Northampton, MA) through extrapolation to the baseline in the regions far from the peaks. The EIS experiments were measured in Buffer IV with the frequency range from 1 Hz to 10 kHz. 3. Results and discussion 3.1. Design and characterization of the sensor As shown in Scheme 1, the sensing platform was composed of four elements: C1eAu electrode, P3-AuNPs, linker DNA (L2) which contained two complementary parts to hybridize with C1 and P3 respectively, two hairpin DNAs H4 and H5. H4 was designed to have a complementary region to H5 and an overhanging 50 - ends to hybridize with P3. In route (a), without methylation, the recognition sites in C1/L2 duplex could be identified and cleaved by HpaII, then further digested by ExoIII. As a result, mimic-HCR could not happen due to the lack of P3-AuNPs, leading to a very low DPV signal. In the presence of M.SssI MTase and SAM, the CpG dinucleotide site in C1/L2 hybridization was methylated, which blocked the cleavage of HpaII. So that mimic-HCR was triggered by P3AuNPs, then subsequently many long nicked dsDNAs formed for

3

RuHex inserting, resulting in a sharp increase in current signal (route b). Thus, the MTase activity assay strategy was constructed based on the difference current signals which is strongly related to MTase activity. The average diameter of the AuNPs used in this research was about 13 nm and had good monodispersity as characterized by TEM (Fig. S2A in supporting information). Only one similar peak at 521 nm for AuNPs and P3-AuNPs was observed in the UVevis absorption spectroscopy (Fig. S2B in supporting information), indicating the modification of P3 kept the dispersivity and the size of the AuNPs. The DNA assembly was characterized by gel electrophoresis (Fig. 1A). As seen in Lanes 1, 2 and 3, there was only one band of each original primer belonging to C1, L2 and P3. The diffuse band appeared in L4 indicating that a larger molecular weight complex was formed by C1, L2 and P3 hybridization. Compared with Lane 1 and 2, Lane 5 had higher band site for C1/L2 forming. Once C1 and L2 complex was cleaved and digested by HpaII and ExoIII, the band disappeared completely (Lane 6). However, the reappearance of diffuse bands in Lane 7 further indicated that the methylated C1/L2 could blocked the cleavage, and did not affect the formation of C1/ L2/P3. These results clearly showed that the three DNA could hybridized with each other, and symmetrical sequence 50 -CCGG-30 in C1/L2 dsDNA could be cleaved or protected, respectively, before and after their methylation successfully. This DNA assembly on gold electrode was confirmed by EIS (Fig. 1B) measurements. With the immobilization of C1, L2 and P3 onto the Au electrode step by step, the charge transfer resistance (Rct) enlarged gradually from 50 U for bare Au electrode (a) to 580 U (b), 970U (c) and 1500 U (d) (Fig. 1B). The charge transfer resistance increased dramatically in the presence of H4 (Fig. 1B, e) and H5 (Fig. 1B, f). The increase of the charge transfer resistance was due to the electrostatic repulsion between the phosphoric acid groups of the DNA backbone and the negatively charged redox indicator ferricyanide increased with the chain of DNA increasing, indicating that each step of DNA hybridization was successful. Especially, mimic-HCR of H4 and H5 formed huge double-helix, which led the impedance to increase sharply. After inserting of RuHex into DNA double-helix, a remarkable drop in the impedance signal was observed (Fig. 2B, g), indicating the electrons could also keep moving quickly in this system due to the strong electrochemical activity of RuHex and the excellent electrical property of Au-NPs. 3.2. Electrochemical behavior of the sensor RuHex has often been used as an electrochemical reporter for various detection of nucleic acids [35,37,42e44] because the redox active RuHex cation could bind with the anionic phosphate backbones of DNA through electrostatic interaction. CV measurement showed a pair of peaks with formal potential of 0.25 V, which were characteristic of the reduction and oxidation of RuHex at DNA coated Au electrode (Fig. S3 in supporting information). This correlated well with the reference [45]. DPV technique was used to sense the feasibility of this proposed signal-amplified sensor for it was much higher sensitive than conventional sweep techniques. When the electrode, regarded as blank electrode, was only modified by C1, a very weak reduction peak was observed due to the small amount of RuHex electrostatic adsorption (Fig. 2, a). The peak changed little after unmethylated C1/L2 on the electrode was cleaved, indicating the sensor had a very low detecting background (Fig. 2, b). In order to test whether the DPV current enlarged sharply only by the means of mimic-HCR, C1eAu electrode was mixed with H5 first so that C1 could open the hairpin of H5 and triggered mimic-HCR of H5 and H4. The reduction current increased to some

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Scheme 1. Schematic representation of the preparation of sensor and signal amplification strategy.

Fig. 2. DPV response of different modification on the Au electrode. (a) C1 modified Au electrode, C1/L2 before (b), and after methylation (c) C1/L2 triggered H4, H5 mimicHCR in absence of P3-AnNPs, (d) P3eAu/L2/C1 modified Au electrode, (e) P3eAu/L2/ C1 triggered H4, H5 mimic-HCR. All electrodes were incubated with Hap II and Exo III.

Fig. 1. (A) EB-stained 12% polyacrylamide gel electrophoresis (PAGE) image. Lane 0, middle range DNA ladder; Lane 1, 10 mM C1; Lane 2, 10 mM L2; Lane 3, 10 mM P3; Lane 4, 5 mM C1þ5 mM L2 þ 5 mM P3; Lane 5, 5 mM C1 þ 5 mM L2; Lane 6, Lane 5 after treatment with Hap II þ Exo III; and Lane 7, methylated Lane 5 after treatment with Hap II and Exo III, then þ5 mM P3. (all the samples were mixed and incubated at 37  C for 1 h); (B) EIS of (a) bare Au electrode, (b) C1 modified Au electrode, (c) L2/C1 modified Au electrode, (d) P3eAu/L2/C1 modified Au electrode, (e) H4 coupled with P3eAu/L2/C1 modified Au electrode, (f) H5/H4 coupled with P3eAu/L2/C1 modified Au electrode and (g) is (f) after being inserted with RuHex.

extent, indicating a little increase RuHex binding in H4/H5 (Fig. 2, c). When P3-AuNPs were modified on methylated C1/L2- Au electrode without HCR of H5 and H4, the current increased a little (Fig. 2, d). The reason might be that Au-NPs accelerated the electrons transfer rate, though there was no more RuHex binding. By contrast, an obvious strong peak was obtained after methylated C1/ L2-electrode was coated by C1/L2/P3-AuNPs and further incubated with H4 and H5, because a single AuNP was loaded with plenty of P3 and many mimic-HCR were triggered to form many huge dsDNAs. Consequently, a large amount of RuHex was introduced onto the electrode surface to enlarge the signal by nearly 4.5 times

(Fig. 2, e) compared with the fabricated way in (c) and (d). These results indicated that the prepared biosensor could acquire amplified DPV signals for the sensitive and specific evaluation of M.SssI MTase activity. 3.3. Optimization of the detection conditions Some detection conditions were optimized in order to obtain the best performance. First of all, the concentration of probe attached on the Au electrode had the most significant influence on the biosensor assembly. Various C1 concentrations from 0.1 mM to 1.2 mM were studied. As shown in Fig. 3A, DPV peak current increased rapidly as the concentration of C1 increased from 0.1 to 0.6 mM, and then further increasing the concentration of C1 led to dramatic decrease in the peak current. The reason might be that too much C1 would hinder the stretching of DNA strands, leading to the decteased efficiency of C1's modification on the electrode. Thus, 0.6 mM was chosen as the optimal C1 concentration. Then, the methylation time was another important parameter for the performance of the biosensor. The electrochemical signals were analyzed with the methylation reaction time ranging from 15 to

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Fig. 3. (A) DPV peak current observed under different C1 concentrations: 0.1, 0.3, 0.6, 0.9 and 1.2 mM; (B) different reaction time of M. SssI MTase: 0.15, 30, 45, 60, 80, 100, 120, 150, and 180 min. 10 mM PB (pH 7.4) containing 100 mM KCl and 5 mM RuHex was used as the buffer. The error bars represent the standard deviation of three measurements.

180 min, and the results were plotted in Fig. 3B. Apparently, the signals increased dramatically from 0 to 90 min and leveled off when the time was prolonged to 120 min. 2 h was chosen hence as the optimal reaction time for CpG methylation in this experiment.

3.4. Detection of M.SssI MTase activity by using the sensor

Fig. 4. DPV response of the sensor treated with different concentrations of M. SssI MTase (a) 0, (b) 0.01, (c) 0.05, (d) 0.1, (e) 0.5, (f) 1, (g) 5, (h) 10, (i) 20, (j) 40, and (k) 60 U mL1. inset: The linear relationship between peak current (mA) and the logarithm of M. SssI MTase concentration ranging from 0.05 to 10 U mL1. The error bars represented the standard deviation of three repetitive measurements in the experimental methods.

Under the optimal conditions, the DPV intensity gradually increased with the increasing concentration of M.SssI MTase (Fig. 4). The inset plot revealed a good linear relationship between DPV intensity and the logarithm of the concentration of M.SssI MTase in the range of 0.05e10 U mL1 with the linear correlation coefficient of 0.994. The detection limit (LOD) was calculated to be 0.03 U mL1 (in terms of the rule of 3 times standard deviation over the blank response), which is much lower than our previously reported method [30] where the DPV response of RuHex amplified by mimic-HCR was over ten times higher than that of polyaniline based only on DNAzyme. The comparison of various analytic performances was list in Table 2. The high sensitivity of the presented work was achieved by signal amplification strategy in a hybridization event and enhancement of the electrons transfer rate with the help of nanostructured materials. The assay precision was examined from the slopes of calibration plots obtained from five independent detection systems. The relative standard derivation (RSD) of these slopes is 4.6%. These results suggest the method can be used for quantitatively analyzing M.SssI MTase activity conveniently and efficiently.

Table 2 Comparison of analytic performance with previously reported methods. Strategy

Amplification assisted by nano-materials

DL (U mL1)

Analytical range (U mL1)

Refs.

Colorimetric assay Fluorescence

No Yes No

0.4 0.03 0.8

0.8e24 0.1e100 0.8e40

16 19 20

Yes No No Yes Yes

0.03 0.52 0.27 0.035 0.0042

0.1e20 1e10 0.5e150 0.1e50 0.01e150

24 23 25 26 33

e

Yes Yes

0.02 0.05

0.075e30 0.1e450

27 29

e

No

0.12

0.5e60

30

e

No

0.28

0.28e50

31

e

No

0.18

0.25e10

45

e

Yes

0.03

0.05e10

this work

e Electrochemiluminescence Chemiluminescence Circular dichroism Photoelectrochemistry e Electrochemical method

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various cancer cells [46,47]. All the inhibition experiments were carried out in the same conditions as that for M.SssI MTase activity assay. The relative activity of the M.SssI MTase decreased gradually with the increasing dose of inhibitors at first, and then tended to level off (Fig. 6), which demonstrated the inhibition was significant dose-dependent and could reach equilibrium at a certain inhibitor concentration. We also noticed that the IC 50 value acquired from Fig. 6 was about 67.7 mM and 244 mM for 5-Aza and procaine, respectively, demonstrating 5-Aza exhibited the higher inhibition efficiency. Their different structure between 5-Aza and procaine may explain this well because 5-Aza is nucleoside analogs which can incorporate into DNA leading to higher demethylating effects. Procaine, however, is a non-nucleoside inhibitor with less toxic effects [48]. 4. Conclusions

Fig. 5. Histogram for the specificity of the biosensor. Both M. SssI and Dam MTase are 50 U mL1.

In summary, a sensitive and specific electrochemical assay was proposed to detect the methyltransferase activity and inhibition by employing signal amplification of mimic-hybridization chain reaction strategy to enhance the assay sensitivity and using endonuclease HpaII and exonuclease III to improve the selectivity. Taking advantage of the mimic-HCR amplified strategy assisted by Au-NPs, the detection limit is as low as 0.03 U mL1 which is superior to most reported strategies. Furthermore, the assay has a good specificity and selectivity, and has the ability to evaluate and screen the inhibitors of M.SssI MTase. Therefore, this detecting strategy holds great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Acknowledgments The project was supported by National Natural Science Foundation of China (Grant No. 21475020 and 21375014). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.aca.2016.05.044. References

Fig. 6. Inhibition of different concentrations of 5-Aza (-) and procaine ( ) on M. SssI MTase activity.

Owing to the specific site recognition of MTase toward its substrate, the proposed sensing system was also performed to discriminate M.SssI MTase from other MTase such as Dam MTase. As shown in Fig. 5, 50 U mL1 of the M.SssI MTase produced a very high DPV current, while the same concentration of Dam MTase produced the same negligible DPV signal as that of the control. This indicated that the proposed strategy based on mimic-HCR and the cleavage function of HpaII and ExoIII has an excellent selectivity for M.SssI MTase activity measurement. 3.5. M.SssI MTase activity inhibition assay Furthermore, this strategy is of interest for screening M.SssI MTase inhibitor. In this case, 5-Aza and procaine were chosen as inhibitor models in subsequent tests because they were previously reported to be anticancer drugs by inhibiting DNA methylation in

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Please cite this article in press as: L. Zhang, et al., Sensitive electrochemical assaying of DNA methyltransferase activity based on mimichybridization chain reaction amplified strategy, Analytica Chimica Acta (2016), http://dx.doi.org/10.1016/j.aca.2016.05.044