Label-Free Sensitive Detection of DNA Methyltransferase by Target

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Label-Free Sensitive Detection of DNA Methyltransferase by TargetInduced Hyperbranched Amplification with Zero Background Signal Yan Zhang, Xin-yan Wang, Qianyi Zhang, and Chun-yang Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03490 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on October 30, 2017

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Analytical Chemistry

Label-Free Sensitive Detection of DNA Methyltransferase by Target-Induced

Hyperbranched

Amplification

with

Zero

Background Signal Yan Zhang,†,§ Xin-yan Wang,†,§ Qianyi Zhang,‡,§ and Chun-yang Zhang*,† †

Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in

Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China. ‡

Nantou High School Shenzhen, Shenzhen, 518052, China

* Corresponding author. Fax: +86 0531-82615258; Tel.: +86 0531-86186033; E-mail: [email protected].

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ABSTRACT: DNA methyltransferases (MTases) may specifically recognize the short palindromic sequences and transfer a methyl group from S-adenosyl-L-methionine to target cytosine/adenine. The aberrant DNA methylation is linked to the abnormal DNA MTase activity, and some DNA MTases have become promising targets of anticancer/antimicrobial drugs. However, the reported DNA MTase assays often involve laborious operation, expensive instruments and radio-labeled substrates. Here, we develop a simple and label-free fluorescent method to sensitively detect DNA adenine methyltransferase (Dam) on the basis of terminal deoxynucleotidyl transferase (TdT)-activated Endonuclease IV (Endo IV)-assisted hyperbranched amplification. We design a hairpin probe with a palindromic sequence in the stem as the substrate and a NH2-modified 3′ end for the prevention of non-specific amplification. The substrate may be methylated by Dam and subsequently cleaved by DpnI, producing three single-stranded DNAs, two of which with 3′-OH termini may be amplified by hyperbranched amplification to generate a distinct fluorescence signal. Because high exactitude of TdT enables the amplification only in the presence of free 3′-OH termini and Endo IV only hydrolyzes the intact apurinic/apyrimidinic sites in double-stranded DNAs, zero background signal can be achieved. This method exhibits excellent selectivity and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61 × 10-6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in disease diagnosis and drug development.

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INTRODUCTION DNA methylation plays important role in cell proliferation, senescence, and gene transcription. 1

The covalent addition of a methyl group to the adenine/cytosine residues of the target is

catalyzed by DNA methyltransferases (MTase) with S-adenosyl-L-methionine(SAM) as the methyl donor.2,3 The DNA methylation positions are usually at N-6 of adenine and C-5/N-4 of cytosine.4 Both excessive methylation (hypermethylation) and deficient methylation (hypomethylation) have been identified in a variety types of cancers, such as breast cancer,5 ovarian cancer 6 and lung cancer.7 In many cases, the aberrant DNA methylation patterns have been associated with the abnormal DNA MTase activity,8-11 and thus sensitive detection of DNA MTase activity is of great importance to the early clinical diagnosis.12 A variety of methods have been developed for DNA MTase assay, such as radioactive assay,13 high-performance liquid chromatography (HPLC),14 immunochemical approaches15,16 and gel electrophoresis.17 But most of them require radio-labeled substrates,13 complicated instrumentation14 and special antibodies,15,16 laborious and time-consuming operation.17 Alternatively, a series of new methods have been developed for DNA MTase assay, such as colorimetry,18-20 fluorometry,21-25 and bioluminescence assays.26 However, these methods usually

involve

tedious

nanoparticle

preparation,18-20

double

labels

with

a

fluorophore/quencher pair21-24 and long analysis time.26 Due to their distinct advantages of low-cost, fast response and miniaturization, electrochemical methods have been widely applied for DNA methylation assay,16,27,28 but they require tedious surface modification of electrodes. To improve the detection sensitivity, a variety of nucleic acid amplification approaches have been introduced, including strand displacement amplification (SDA),29 3



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primer generation exponential isothermal amplification (PG-EXPAR)30 and rolling circle amplification (RCA).31,32 Despite the improved sensitivity, these approaches usually require the specific recognition sequences for nicking enzyme and complex probe design.29-32 In addition, non-specific amplification may occur due to involvement of thermophilic DNA polymerase.33,34 Therefore, a simple method with high sensitivity and good selectivity is highly desirable for DNA MTase assay. In this research, we develop a simple and label-free fluorescent method to sensitively detect DNA MTase on the basis of TdT-activated Endo IV-assisted hyperbranched amplification. TdT is a special DNA polymerase that catalyzes the repetitive addition of deoxynucleotide triphosphates (dNTPs) to the 3’-hydroxy terminus (3′-OH) of DNA molecules without the requirement of any DNA templates.35 Endo IV is an endonuclease that may hydrolyze any intact apurinic/apyrimidinic (AP) sites in double-stranded DNAs (dsDNAs). In comparison with other nucleic acid amplification strategies, the TdT-activated enzyme-assisted hyperbranched amplification has significant advantages of simple reaction process without the requirement of either DNA templates or special design of specific restriction enzyme recognition sequences. This assay shows good specificity with zero background signal and high sensitivity with a limit of detection of 0.003 U/mL for pure Dam and 9.61×10-6 mg/mL for Dam in E. coli cells. Moreover, it can be used to screen the Dam inhibitors, holding great potentials in drug discovery and early clinical diagnosis.

EXPERIMENTAL SECTION Materials. The oligonucleotides (Table 1) were obtained from Shanghai Sangon Biological 4


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Engineering Technology & Services Co. Ltd. (Shanghai, China). DNA adenine methyltransferase (Dam), DpnI, terminal transferase, S-adenosylmethionine (SAM) and Endonuclease IV (Endo IV) were obtained from New England Biolabs (Ipswich, MA). Deoxyadenosine triphosphate (dATP) was purchased from TaKaRa Biotechnology Co. Ltd. (Dalian, China). SYBR Gold was purchased from Life Technologies (Carlsbad, CA). The 5-fluorouracil and other chemicals with analytical grade were purchased from Sigma-Aldrich Co. (St. Louis, MO). All solutions were prepared with ultrapure water (Millipore, Milford, MA). Table 1. Sequences of the Oligonucleotides α note hairpin substrate

sequence (5′-3′) GAA GGA TCT TCT CGA CTT GCT GAA GAT CCT TCT TAA T-NH2

primer

TCT TCT CGA CTT GCT GAA GA

assistant probe

TTT TTT TTT TTT TTT TTT TTX TTT TT-NH2

α

The Dam recognition site in the hairpin substrate is marked by boldface. The X in the

assistant probe indicates the abasic site mimic.

Culture of Bacterial Cells. The colony of JM110 (DAM negative) and GW5100 (DAM positive) E. coli cells were inoculated into 3 mL of liquid medium (5 g/L yeast extract, 10 g/L Trypton, 10 g/L NaCl), respectively, and incubated at 37 °C in a rotary shaker at 250 rpm for 12 h. Then 100 µL of cell suspension was subsequently added into 3 mL of medium and incubated for 2.5 h. Subsequently, the cell suspension was centrifuged at 5000 rpm to obtain a 5



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cell pellet. After washing twice with pure water, the E. coli cells were lysed by using RIPA lysis buffer, and the protein contents were determined by using the Bradford Protein Assay Kit. The cell lysate was flash frozen and stored at −80 °C for further use. Detection of Dam Activity. The methylation and cleavage of hairpin substrate were performed in 200 µL of reaction mixture containing 0.5 µM hairpin probe, 160 µM SAM, 10 U of DpnI, 1× Dam reaction buffer (10 mM EDTA, 50 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5) and various-amount Dam for 2 h at 37 ℃. The reaction was terminated by inactivation for 20 min at 80 ℃. Hyperbranched amplification was performed in 30 µL of reaction system containing 1× TdT buffer (10 mM Mg(Ac)2, 20 mM Tris-Ac, 50 mM KAc, pH 7.9), 0.25 Mm CoCl2, 1× SYBR Gold, 0.65 µM assistant probe, 1 mM dATP, 4 U of Endo IV, 8 U of TdT and 4 µL of methylation products. The typical polymerization experiment was performed on Bio-Rad CFX 96 Real-Time PCR instrument (Bio-Rad, USA) at 37 °C for 100 min. The fluorescence signals were detected at intervals of 30 s. Gel electrophoresis. The 12% nondenaturating polyacrylamide gel electrophoresis (PAGE) was performed in 1× TBE buffer (9 mM boric acid, 9 mM Tris-HCl, 0.2 mM EDTA, pH 7.9) using 1× SYBR Gold as the stain at 110 V constant voltage for 50 min at room temperature. Selectivity of Dam Assay. For selectivity assay, we employed BSA and M.SssI MTase as the nonspecific enzymes. The experiment was performed with 20 U/mL nonspecific enzyme using the procedure described above. Inhibition Assay. To investigate the influence of 5-fluorouracil on the activity of Dam, various-concentration 5-fluorouracil, 0.5 µM hairpin probe and 1× Dam buffer (10 mM EDTA, 50 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5) were incubated for 15 min at 6


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37 °C. Then 20 U/mL Dam, 160 µM SAM and 50 U/mL DpnI were added to the mixture and incubated for 2 h at 37 °C. The reaction was terminated by inactivation at 80℃ for 20 min. The Dam activity was measured using the procedure described above, and its relative activity (RA) was calculated by equation 1:

RA =

Fi − F 0 × 100% Ft − F 0

(1)

where F0 is the fluorescence intensity when Dam is absent, and Ft is the fluorescence intensity when 20 U/mL Dam is present, and Fi is the fluorescence intensity when both 5-fluorouracil and Dam are present.

RESULTS AND DISCUSSION Principle of DNA MTase Assay. In prokaryotic organisms, Dam can methylate the N6-adenine in the 5′-G-A-T-C-3′ sequence and regulates various cellular processes, such as transcription of a certain gene, DNA mismatch repair and DNA replication.36-38 The principle of DNA MTase assay is illustrated in Scheme 1 with Dam as the model DNA MTase. We designed a hairpin DNA probe whose stem contains 5′-G-A-T-C-3′ as the substrate. In order to prevent TdT-activated non-specific amplification, we modified the 3′ termini of the hairpin probe and the assistant probe with NH2. Upon treatment with Dam, the 5′-G-A-T-C-3′ is methylated to 5′-G-mA-T-C-3′ for the achievement of a methylated hairpin DNA probe. The subsequent cleavage of methylated hairpin DNA probe by endonuclease DpnI 39 releases three single-stranded DNA (ssDNA) fragments, two of which contain free 3′-OH termini. In the presence of TdT, multiple dATPs may be sequentially added to the free 3′-OH termini of ssDNAs to obtain the A-rich sequences. The NH2-modified assistant probe with both T-rich 7



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sequence and an AP site may hybridize with the resultant A-rich sequence to form a stable dsDNA. The AP site in dsDNA may be catalyzed by Endo IV, resulting in the break of assistant probe and the generation of free 3′-OH termini. The new DNA fragments with free 3′-OH termini may initiate new TdT-mediated extension reaction to form a much longer A-rich sequence. Notably, the excess assistant probes may hybridize with the A-rich sequence to trigger new cycles of cleavage-extension, inducing hyperbranched amplification for generation of a large number of DNA fragments which may produce a distinct fluorescence with SYBR Gold as the indicator. While in the absence of Dam, neither TdT-mediated extension nor Endo IV-mediated cleavage of assistant probes can be initiated, and no distinct fluorescence is observed.

Scheme 1. Principle of Dam assay based on TdT-activated Endo IV-assisted hyperbranched amplification a a

In the presence of Dam, the cleavage of methylated hairpin DNA probe by DpnI generates

three ssDNA fragments. Two ssDNAs with free 3′-OH termini may be subsequently amplified 8


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by TDT-activated Endo IV-assisted hyperbranched amplification to generate a distinct fluorescence signal with SYBR Gold as the indicator.

We performed nondenaturating polyacrylamide gel electrophoresis (PAGE) to investigate the methylation process of Dam (Figure 1A). In the absence of either Dam or DpnI, only one band of 20 bp is observed (Figure 1A, lanes 2-3), suggesting no occurrence of cleavage event. In the presence of both Dam and DpnI, a new 20-nt band is observed (Figure 1A, lane 1) as a result of methylation and cleavage reactions. We further performed PAGE analysis to investigate the effect of incubation time of TdT and Endo IV upon the length of amplification products. In the reported TdT-catalyzed enzymatic polymerization, the number average degree of polymerization increases with the amplification reaction time.40 Our result indicates that the length of products increases with the amplification reaction time from 0 to 120 min (Figure 1B), consistent with the result of previous research.40 To shorten the assay time, we used 100 min as the amplification reaction time. To verify the TdT-activated Endo IV-assisted hyperbranched amplification, we used PAGE to monitor the amplification products at the reaction time of 100 min. When both Dam and DpnI are present, a well-defined band of amplified products is observed (Figure 1C, lane 1), indicating the production of large amounts of DNA fragments as a result of hyperbranched amplification. In contrast, no band of amplified products is observed in the control group without Dam (Figure 1C, lane 2). To further confirm the TdT-activated Endo IV-assisted hyperbranched amplification, we performed real-time fluorescence measurement as well (Figure 1D). When both Dam and Dpn I are present, the fluorescence signal enhances linearly 9



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with time (Figure 1D, red line). While in the control group without Dam, zero background signal is observed (Figure 1D, black line). The achievement of zero background signal may be ascribed to the modification of the 3′ termini of hairpin probe and assistant probe with NH2 which may efficiently prevent the cleavage of hairpin DNA probe by DpnI in the absence of Dam.

Figure 1. (A) PAGE monitoring of the methylation and cleavage reactions under different conditions. Lane M, DNA ladder marker; lane 1, 0.5 µM hairpin substrate + 20 U/mL Dam + 50 U/mL DpnI; lane 2, 0.5 µM hairpin substrate + 50 U/mL DpnI; lane 3, 0.5 µM hairpin substrate + 20 U/mL Dam. (B) Influence of amplification reaction time upon the length of products. All the amplification reactions are performed in the presence of 100 nM primer and 650 nM assistant probe. Lane M, DNA ladder marker; lane 1, 0 min; lane 2, 10 min; lane 3, 30 min; lane 4, 60 min; lane 5, 90 min; lane 6, 120 min. (C) PAGE analysis of the 10


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hyperbranched amplification products under different conditions. Lane M, DNA ladder marker; lane 1, 20 U/mL Dam + 50 U/mL DpnI; lane 2, 50 U/mL DpnI. (D) Real-time fluorescence monitoring of hyperbranched amplification in the presence of 20 U/mL Dam + 50 U/mL DpnI (red line) and without Dam (black line).

Optimization of Experimental Conditions. To obtain the best assay performance, we optimized the concentrations of assistant probe and dATP, and the amounts of Endo IV and TdT, respectively. The concentration of assistant probe has a crucial effect upon the amplification efficiency. On the one hand, the low-concentration assistant probe might adversely affect the amplification efficiency, because the hyperbranched amplification efficiency is highly dependent on the capability of assistant probe to hybridize with the A-rich sequence to trigger the cycles of cleavage-extension for the generation of a large number of DNA fragments. But on the other hand, the high-concentration assistant probe might cause the dimerization of assistant probes and adversely affect the amplification efficiency.41 Therefore, we optimized the concentration of assistant probe (Figure 2A), The F-F0 value improves with the increasing concentration of assistant probe, and reaches a plateau at the concentration of 0.65 µM (F is the fluorescence intensity when Dam is present, and F0 is the fluorescence intensity when Dam is absent). Thus, 0.65 µM assistant probe is used in the subsequent experiments. The fluorescence signal relies on both TdT-mediated extension and Endo IV-mediated cleavage of assistant probes, and thus the amounts of Endo IV and TdT should be optimized. We studied the effect of Endo IV on the fluorescence signal with the amount of TdT being 11



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fixed at 8U (Figure 2B). The F-F0 value enhances with the increasing amount of Endo IV, and reaches the maximum value at the amount of 4 U (F is the fluorescence intensity when Dam is present, and F0 is the fluorescence intensity when Dam is absent). We further studied the effect of TdT on the fluorescence signal with the amount of Endo IV being fixed at 4U (Figure 2C). The F-F0 value improves with the increasing amount of TdT from 4 to 8 U, followed by the decrease beyond the amount of 8 U (F is the fluorescence intensity when Dam is present, and F0 is the fluorescence intensity when Dam is absent). Notably, even though the precise mechanism for enzymatic interaction, cooperation and competition with one another remains unclear, we may simply modulate the reaction by changing the amount of TDT polymerase and Endo IV. Based on Figures 2B-2C, we used 4 U Endo IV and 8 U TdT in the subsequent experiments. TdT can catalyze the repetitive addition of dATPs to the 3’-hydroxy terminus of DNA molecules without the requirement of any DNA templates,35 and thus the influence of dATP concentration upon the fluorescence signal should be investigated. Figure 2D shows that the

F-F0 value enhances with the increasing concentration of dATP, and reaches the maximum value at the concentration of 1 mM (F is the fluorescence intensity when Dam is present, and

F0 is the fluorescence intensity when Dam is absent). This may be explained by TdT deactivation at high monomer (dATP) concentration as a result of the incorporation of monomers into the growing polynucleotide chains.40 Based on Figure 2D, we used 1 mM dATP in the subsequent experiments.

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Figure 2. (A) Variance of the F-F0 value with the concentration of assistant probe. (B)Variance of the F-F0 value with the amount of Endo IV at a fixed amount of TdT (8U). (C)Variance of the F-F0 value with the amount of TdT at a fixed amount of Endo IV (4U). (D)Variance of the F-F0 value with the dATP concentration. F is the fluorescence intensity when Dam is present, and F0 is the fluorescence intensity when Dam is absent. The Dam concentration is 20 U/mL. The fluorescence intensities are obtained at 100 min. Error bars represent the standard deviation from three independent experiments.

Detection Sensitivity. Under the optimally experimental conditions, we monitored the fluorescence signal generated by different-concentration Dam in real time. As shown in Figure 3A, the fluorescence signal increases linearly in both time-dependent and concentration-dependent manners. The higher concentration of Dam, the more DNA

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substrates being methylated by Dam and cleaved by DpnI, the more free 3′-OH termini being generated and consequently the higher fluorescence signal. In the logarithm scale, the fluorescence intensity exhibits a good linear correlation with the concentration of Dam over 4 orders of magnitude in the range from 0.005 to 40 U/mL (Figure 3B). The corresponding equation is F =2889.8 + 1164.4 log10 C (R2 = 0.991), where F is the fluorescence intensity and C is the Dam concentration (U/mL). The limit of detection is evaluated to be 0.003 U/mL. The sensitivity of this assay has enhanced by 1 order of magnitude as compared with those of exonuclease-mediated target recycling-based fluorescent assay (0.01 U/mL)24, nicking enzyme-assisted

signal

amplification-based

fluorescent

assay

(0.06

U/mL)42

and

transcription-mediated cyclic signal amplification-based fluorescent assay (0.015 U/mL),23 by 2 orders of magnitude as compared with those of DNAzyme-mediated signal amplification-based fluorescent assay (0.4 U/mL)22 and colorimetric assay (0.25 U/mL).29 Importantly, the proposed method is extremely simple without the requirement of any specific recognition sequences for nicking enzyme, and it exhibits excellent specificity with zero background signal in the absence of target Dam. The enhanced sensitivity can be ascribed to three factors: (1) highly efficient TdT-activated Endo IV-assisted hyperbranched amplification induces enhanced fluorescence signal; (2) high exactitude of TdT enables the occurrence of amplification only in the presence of free 3′-OH termini and Endo IV can only hydrolyze the intact AP sites, resulting in zero background signal; and (3) the modification of 3′ termini of hairpin probe and assistant probe by NH2 efficiently prevents the TdT-activated non-specific amplification.

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Figure 3. (A) Real-time fluorescence curves in response to different-concentration Dam. (B) The fluorescence intensity shows a linear correlation with the logarithm of Dam concentration. The fluorescence intensities in Figure 3B are obtained at 100 min. Error bars represent the standard deviation from three independent experiments.

Detection Selectivity. For selectivity assay, we employed BSA and M.SssI MTase as the nonspecific enzymes. BSA is an irrelevant protein, and M.SssI MTase is a methyltransferase that can specifically methylate the cytosine residues of 5′-C-G-3′ in dsDNA. When Dam is present, the fluorescence signal enhances linearly in a time-dependent manner (Figure 4A, red line). In contrast, no fluorescence signal is detected in the presence of BSA (Figure 4A, pink line), M.SssI MTase (Figure 4A, blue line), and the control group with only buffer (Figure 4A, black line). Moreover, the fluorescence intensity in response to Dam is much higher than those in response to M.SssI MTase, BSA and the control group (Figure 4B).This may be explained by the fact that the 5′-G-A-T-C-3′ can only be methylated by Dam instead of BSA and M.SssI MTase. These results clearly demonstrate that this method possesses excellent selectivity toward Dam.

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Figure 4. (A) Real-time fluorescence curves in response to Dam (red line), M.SssI MTase (blue line), BSA (pink line), and the control group with only buffer (black line), respectively. (B) Measurement of fluorescence intensity in response to Dam, M.SssI MTase, BSA and the control group with only buffer. The fluorescence intensities in Figure 4B are obtained at 100 min. The Dam concentration is 20 U/mL, and the M.SssI MTase concentration is 20 U/mL, and the BSA concentration is 20 U/mL. Error bars represent the standard deviation from three independent experiments.

Detection of Dam in E. coli Cells. Previous researches demonstrate that the level of Dam in E. coli cells at the exponential growth stage is much higher than that in the stationary stage. 43,44

To demonstrate real sample analysis, we measured the endogenous Dam in E. coli cells at

the exponential growth stage. As shown in Figure 5A, a high fluorescence signal is observed in Dam-positive E. coli of GW5100 (Figure 5A, red column), but no significant fluorescence signal is observed in Dam-negative E. coli of JM110 (Figure 5A, grey column), suggesting that the obtained fluorescence signal is derived from Dam instead of other enzymes in total proteins. Moreover, in the logarithmic scale, a linear correlation is achieved between the

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fluorescence intensity and the total protein concentration of GW5100 over 4 orders of magnitude from 1.5 × 10-5 to 0.15 mg/mL (Figure 5B). The corresponding equation is F = 4892.83 + 972.09 log10 C (R2 = 0.9908), where C represents the concentration of total protein of E. coli cells (mg/mL) and F represents the fluorescence intensity. The limit of detection is experimentally estimated to be 9.61 × 10-6 mg/mL, which is much lower than that of exonuclease-mediated target recycling-based fluorescent assay (5.5 × 10-5 mg/mL)24, suggesting the feasibility of the proposed method for further application in clinical samples.

Figure 5. (A) Measurement of Dam in Dam-positive E. coli of GW5100 and Dam-negative E. coli of JM110. The total protein concentration in GW5100 is 0.1 mg/mL, and the total protein concentration in JM110 is 0.1 mg/mL. (B) The fluorescence intensity shows a linear correlation with the logarithm of total protein concentration of GW5100 in the range from 1.5 × 10-5 to 0.15 mg/mL. The fluorescence intensities are obtained at 100 min. Error bars represent the standard deviation from three independent experiments.

Inhibition Assay. Dam plays important roles in the virulence of a growing list of bacterial

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pathogens, and is a promising target in antimicrobial drug discovery.38 For inhibition assay, we used 5-fluorouracil as the model inhibitor. The 5-fluorouracil does not affect the DpnI activity at the concentrations of less than 10 µM.29 We performed gel electrophoresis analysis and real-time fluorescence measurement to investigate the effect of 5-fluorouracil on the activities of TDT and Endo IV using a synthetic primer with free 3′-OH termini. As shown in Figure 6A, a distinct band of TdT-mediated amplification product is observed in the presence of TDT no mater treatment with 10 µM 5-fluorouracil (Figure 6A, lane 2) or not (Figure 6A, lane 3), with no difference being observed between them, suggesting that 10 µM 5-fluorouracil does not affect the TdT activity. In addition, a distinct band of TdT-activated Endo IV-assisted hyperbranched amplification product is observed in the presence of TDT and Endo IV no mater treatment with 10 µM 5-fluorouracil (Figure 6A, lane 4) or not (Figure 6A, lane 5), with no difference being observed between them, suggesting that 10 µM 5-fluorouracil does not affact the Endo IV activity. Importantly, the fluorescence intensity obtained in the presence of 10 µM 5-fluorouracil (Figure 6B, red column) shows no obvious difference from that obtained in the absence of 5-fluorouracil (Figure 6B, grey column) no matter in the presence of TdT or in the presence of TDT and Endo IV, indicating that 10 µM 5-fluorouracil has no obvious effect on the activities of TDT and Endo IV. These results demonstrate that 5-fluorouracil does not influence the enzymatic activities of DpnI, TdT and Endo IV. Figure 7A shows the effect of 5-fluorouracil on the relative activity of pure Dam. The 5-fluorouracil induces the decrease of Dam relative activity in a concentration-dependent manner. The IC50 value is the inhibitor concentration that induces the decrease of enzyme 18


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activity by 50%. The calculated IC50 value is 1.29 µM, consistent with that obtained by PG-RCA-mediated chemiluminescent assay (1.42 ± 0.07 µM).31 Furthermore, we examined the inhibition effect of 5-fluorouracil upon Dam activity using E. coli cell lysate. As shown in Figure 7B, the fluorescence intensity obtained in the presence of 8 µM 5-fluorouracil (Figure 7B, red column) is much lower than that obtained in the absence of 5-fluorouracil (Figure 7B, grey column), indicating that the Dam activity in E. coli cells can be efficiently inhibited by 5-fluorouracil. These results (Figures 6-7) suggest that this method can be used to screen the Dam inhibitors.

Figure 6. (A) Gel electrophoresis analysis of the effect of 5-fluorouracil upon the activities of TdT and Endo IV. Lane M, DNA marker; lane 1, 67 nM primer; lane 2, 67 nM primer + 650 nM assistant probe + 8 U TdT; lane 3, 67 nM primer + 650 nM assistant probe + 8U TdT + 10 µM 5-fluorouracil; lane 4, 67 nM primer + 650 nM assistant probe + 8 U TdT + 4 U Endo IV; lane 5, 67 nM primer + 650 nM assistant probe + 8 U TdT + 4 U Endo IV + 10 µM 5-fluorouracil. (B) Measurement of fluorescence intensity in the presence of 10 µM 5-fluorouracil (red column) and in the absence of 5-fluorouracil (gray column). The fluorescence intensities are obtained at 100 min. Error bars represent the standard deviation 19



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from three independent experiments.

Figure 7. (A) Inhibition effect of 5-fluorouracil on the activity of pure Dam. (B) Measurement of fluorescence intensity in response to 0.1 mg/mL Dam-positive E. coli cell lysate in the presence (red column) and absence of 5-fluorouracil (gray column). The 5-fluorouracil concentration is 8 µM. The fluorescence intensities are obtained at 100 min. Error bars represent the standard deviation from three independent experiments.

CONCLUSIONS In conclusion, we have demonstrated the development of a simple and label-free fluorescent method for real-time monitoring of DNA MTase activity on the basis of TdT-activated Endo IV-assisted hyperbranched amplification. We modified the 3′ termini of hairpin probe and assistant probe with NH2 to prevent the TdT-activated non-specific amplification. In our design, multiple dATPs can only be incorporated into 3′-OH terminal of DNA by TdT and only intact AP sites in dsDNA can be hydrolyzed by Endo IV. As a result, zero background signal can be achieved in the absence of Dam. The combination of TdT-mediated extension

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with Endo IV-mediated cleavage of assistant probes can induce efficient hyperbranched amplification to produce a large number of DNA fragments, ensuring the high sensitivity of DNA MTase assay. In contrast to the reported fluorescent methods,20-23 this method is label-free and cost-effective without the requirement of any fluorescent modifications. In comparison with the reported isothermal amplification approaches,27-30 this method has significant advantages of simple reaction scheme without the involvement of either multiple enzymes for the preparation of circular template or the specific restriction enzyme recognition sequence for nicking enzyme cleavage. In addition, this assay may be performed in a homogeneous format. This method is very sensitive with a limit of detection of 0.003 U/mL for pure Dam and 9.61×10-6 mg/mL for Dam in E. coli cells. Importantly, this method has the capability of discriminating Dam from other methyltranferases and screening the Dam inhibitors, providing a broad spectrum of therapeutic applications such as disease diagnosis and drug discovery.

AUTHOR INFORMATION Corresponding Author * Fax: +86 0531-82615258. Tel.: +86 0531-86186033. E-mail: [email protected]. Author Contributions §

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT 21



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This work was supported by the National Natural Science Foundation of China (Grant Nos. 21325523, 21527811, 21735003 and 21605098) and the Award for Team Leader Program of Taishan Scholars of Shandong Province, China. We thank Prof. Bi-Feng Yuan at Wuhan University for providing the GW5100 and JM110 E. coli strains.

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For TOC only

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Label-Free Sensitive Detection of DNA Methyltransferase by Target

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