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A loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity
Sensors and Actuators B 244 (2017) 599–605
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity Wanling Cui a , Lei Wang b , Xiaowen Xu a , Yan Wang c,∗∗ , Wei Jiang a,∗ a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, P.R. China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, P.R. China c The 88th Hospital of PLA, 270100 Tai’an, P.R. China
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 6 December 2016 Accepted 3 January 2017 Available online 8 January 2017 Keywords: DNA methyltransferase activity Long stem-loop probe Strand displacement amplification Exponential rolling circle amplification Fluorescent detection
a b s t r a c t DNA methyltransferase (MTase) is a predictive cancer biomarker and drug target, and the sensitive detection of DNA MTase activity is crucial to early cancer diagnosis and therapy. In this work, we developed a loop-mediated cascade amplification strategy for highly sensitive fluorescent detection of DNA MTase activity based on strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Firstly, we designed a long stem-loop probe (LSLP) which contains a methylation site for DNA MTase recognition, a long stem for ensuring the stability of probe, and a loop for initiating subsequent amplification. The loop and part of stem of LSLP acted as a trigger strand for subsequent signal output process. And the trigger strand was fully enclosed in loop of LSLP by the long stem, avoiding the nonspecific amplification caused by the leakage of probe. The LSLP was methylated by DNA MTase and then was specifically cleaved by DpnI endonuclease, producing a trigger strand. Under the synergetic action of polymerase and nicking enzyme, the trigger strand initiated SDA, producing many primers. The produced primers initiated ERCA, synthesizing numerous G-quadruplex sequences. The G-quadruplex sequences interacted with N-methylmesoporphyrin IX, obtaining an enhanced fluorescent signal. The method could detect as low as 8.1 × 10−5 U/mL DNA MTase. Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase activity. These results show that our system has a great potential in early cancer diagnosis and therapy. © 2017 Elsevier B.V. All rights reserved.
1. Introduction DNA methyltransferase (MTase) is an epigenetic modification enzyme, which plays a vital role in many important biological processes, including the regulation of gene expression, developmental regulation and genomic imprinting [1–3]. It catalyzes DNA methylation by the covalent addition of methyl groups to adenine or cytosine in the corresponding recognition sequences [4–7]. Studies have shown that abnormal DNA MTase activity is closely related to the initiation and progression of diseases such as cancer [8–10]. Accordingly, DNA MTase activity has been widely seen as a predictive cancer biomarker and drug target [11–14]. Therefore, the sensitive detection of DNA MTase activity is of significant importance for DNA MTase related cancer diagnosis and therapy.
∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (W. Jiang). http://dx.doi.org/10.1016/j.snb.2017.01.013 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Traditionally, radioactive labeling [15], gel electrophoresis [16], and high-performance liquid chromatography [17,18] are used for the detection of DNA MTase activity. In addition to the above methods, many new strategies for the detection of DNA MTase activity have been developed to improve the detection sensitivity and specificity, including electrochemical [19–21], chemiluminescent [22,23], colorimetric [24,25] and fluorescent methods [26–29]. Among them, fluorescent methods as powerful bioanalytical tools for DNA MTase activity assay have received the widespread attention. In general, fluorescent methods have been established by using double-stranded DNA (dsDNA) probe or hairpin probe with overhang to achieve target recognition and signal transduction [30–34]. The signal transduction strand is partially enclosed in the dsDNA probe or stem of hairpin probe. Under the recognition action of DNA MTase, the dsDNA probe or hairpin probe releases a signal transduction strand and then the signal transduction strand triggers the subsequent signal output process. The partially enclosed manner leads to nonspecific amplification caused by leakage of probe, resulting in false positive signal. However, the competing
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hybridization between dsDNA probe or hairpin probe and report probe happens in the absence of DNA MTase [35,36], which may causing the nonspecific background amplification, compromising detection sensitivity and accuracy. In order to solve those problems, we developed a loop-mediated cascade amplification strategy for highly sensitive fluorescent detection of DNA MTase activity based on strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Firstly, we designed a long stem-loop probe (LSLP) which contains a methylation site for DNA adenine methylation (Dam) MTase recognition, a long stem for ensuring the stability of probe, and a loop for initiating subsequent amplification. The loop and part of stem of LSLP acted as a trigger strand for subsequent signal output process. And the trigger strand was fully enclosed in loop of LSLP by the long stem, avoiding the nonspecific amplification caused by the leakage of probe. The LSLP was methylated by DNA MTase and then was specifically cleaved by DpnI endonuclease (DpnI), producing a trigger strand. Then, the trigger strand initiated SDA and ERCA, synthesizing large numbers of G-rich sequences. Finally, the G-rich sequences selectively interacted with N-methylmesoporphyrin IX (NMM), obtaining an enhanced fluorescent signal. By effective combination of LSLP design and cascade amplification strategy, this method could detect as low as 8.1 × 10−5 U/mL DNA MTase, which is superior or comparable to that of the reported literature. Moreover, this method could well distinguish Dam MTase from other MTases (AluI, HhaI, M.SssI and HaeIII MTases). Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase activity using gentamycin, benzylpenicillin and 5–fluorouracil as model. These results shows that our system has a great potential in early cancer diagnosis and therapy. 2. Experimental section 2.1. Materials and apparatus Synthetic DNA oligonucleotides were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences were shown in Table S1 in the Supporting Information. DNA adenine methylation (Dam), HaeIII, HhaI, M.SssI, and AluI MTase, DpnI endonuclease, S-adenosyl-l-methionine (SAM), Klenow Fragment (3 –5 exo-) polymerase, T4 DNA ligase, and Nt.BbvCI were obtained from New England Biolabs (Ipswich, MA, USA). Gentamycin, benzylpenicillin, 5–fluorouracil were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and used as received. Phi29 DNA polymerase was from Fermentas (Lithuania). All other reagents used in this work were analytical grade and used without further purification or modification. All solutions were prepared in ultrapure water (> 18.25 M cm) from a Millipore Milli-Q water purification system. The fluorescence spectra were recorded using Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan). The emission spectra were collected from 560 to 690 nm and excitation wavelength was set at 399 nm. The fluorescence emission intensity was measured at 618 nm. Both excitation and emission slit widths were all set at 10.0 nm and the photomultiplier tube voltage was 700 V. 2.2. Assay of Dam MTase activity In the typical methylation and cleavage experiments, 20 nM LSLP, 160 M SAM, 2 U DpnI and various amounts of Dam MTase were put into 10 L of methylase buffer (50 mM NaCl, 10 mM TrisHCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.5) and incubated at 37 ◦ C for 2 h. Then, 40 nM hairpin probe, 1 U Klenow Fragment (3 –5 exo-), 0.6 mM dNTPs, 2 U Nt.BbvCI and 1 × CutSmart (50 mM KAc, 20 mM Tris-HAc, 10 mM Mg (Ac) 2 , 100 g/mL BSA, pH 7.9) were
added. After the incubation at 37 ◦ C for 30 min, the reaction mixture was heated at 85 ◦ C for 20 min to deactivate the enzymes. After that, 120 U T4 DNA ligase, 800 nM padlock probe and 1 × T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) were put into the above reaction mixture and kept at 37 ◦ C for 1 h to obtain the circular probe. Subsequently, 1 mM dNTPs, 3 U Phi29 DNA polymerase, 4 U Nt.BbvCI and 1 × CutSmart were added. The reaction was carried out at 37 ◦ C for 3 h, and then terminated by heating to 75 ◦ C for 20 min. Finally, 200 M KCl and 3 M NMM were added and incubated at 37 ◦ C for 30 min, followed by the fluorescence measurement. 2.3. Gel electrophoresis to study methylation and cleavage reactions The gel electrophoresis was used to determine whether or not the methylation and cleavage reactions were carried out, by separating the DpnI-cleaved products from the LSLP. The different samples were analyzed by putting them on a 15% nondenaturating polyacrylamide gel electrophoresis (PAGE). The electrophoresis was performed in 1 × Tris-borate-EDTA (TBE) buffer (89 mM Tris, 89 mM Boric Acid, 2.0 mM EDTA, pH 8.3) at 30 mA for 1.5 h, followed by ethidium bromide staining. The gels were photographed under UV imaging system (Bio-RAD Laboratories Inc., USA). 2.4. Effect of inhibitors on Dam MTase activity In inhibition assay, we chose three inhibitors (gentamycin, benzylpenicillin, and 5–fluorouracil) to investigate the inhibition effect of inhibitors on Dam MTase activity. The inhibition experiment was performed in same procedures as that mentioned above for Dam MTase activity assay, except for needing add various concentrations of the inhibitors. The inhibition effect of inhibitors was evaluated by the relative activity of Dam MTase, which could be expressed by the equation: relative activity = (F2 –F0 )/(F1 –F0 ), where F2 and F1 were fluorescent intensity of 8 U/mL Dam MTase with or without inhibitor respectively, and F0 was fluorescent intensity without Dam MTase. Our assay system contained auxiliary enzymes (DpnI, Klenow Fragment (3 –5 exo-) polymerase, T4 DNA ligase, and Nt.BbvCI, and Phi29 DNA polymerase). Control experiments were used to investigate whether or not inhibitors had effect on the activities of those enzymes. The methylation experiment was carried out at 37 ◦ C for 3 h to obtain the absolutely methylated probe, and then was heated at 65 ◦ C for 20 min to terminate the reaction. Subsequently, 1 M inhibitor and the other reagents were added to investigate inhibition effect of inhibitor on auxiliary enzymes. 3. Results and discussion 3.1. Principle of cascade amplification strategy for Dam MTase activity assay Scheme 1 outlined the design principle of sensing system for DNA MTase activity assay. Due to the same recognition site of Dam MTase and DpnI, they were respectively selected as the model DNA MTase and methylation-sensitive restriction endonuclease to verify the feasibility of the method. In the presence of Dam MTase, Dam MTase recognized and methylated the LSLP to form the methylated LSLP containing the sequences of 5 -GmATC-3 . Then, DpnI specifically cleaved the methylated LSLP between the two methyladenines into two parts. One part was a dsDNA, and the other one was a new hairpin probe. Under the experimental conditions, the new hairpin probe was unstable and underwent a conformational change to single-stranded DNA (ssDNA), due to the low melting temperature (Tm ) value. The ssDNA hybridized with the overhang
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Scheme 1. Experimental principle of the loop-mediated cascade amplification strategy for DNA MTase activity detection through SDA and ERCA. In long stem-loop probe, the green segment is the recognition site of DNA MTase, the purple loop segment is the trigger sequence that can trigger SDA, and the blue stem segment is used to close the trigger sequence. In hairpin probe, the purple segment is complementary to the trigger sequence in long stem-loop probe, and the light blue segment is the recognition site of Nt.BbvCI nicking endonuclease. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
of hairpin probe and acted as a trigger strand to trigger a polymerization reaction in the presence of Klenow Fragment polymerase and dNTPs, yielding a duplex strand with a full recognition site of Nt.BbvCI nicking enzyme. After the cleavage of Nt.BbvCI nicking enzyme, a new replication site for next polymerization reaction was produced. Under the synergetic action of polymerase and nicking enzyme, such repeated polymerization and nicking reactions happened, releasing many primers. Every one of the released primers could hybridize with the padlock probe in the presence of T4 DNA ligase to obtain a circular probe, which contained three recognition sites of Nt.BbvCI, two complementary sequences of G-rich sequence and a binding region of primer. Subsequently, by the addition of Phi29 DNA polymerase, the primer triggered RCA, synthesizing a long DNA product containing many concatenated sequences. Then, the long DNA product hybridized with multiple padlock probes, forming stable dsDNAs with multiple recognition sites of Nt.BbvCI. Next, Nt.BbvCI cleaved the stable dsDNAs into short dsDNAs. Due to the low melting temperature value of short dsDNAs, large numbers of G-rich sequences and primers that used for next RCA were produced. Through the synergetic action of Phi29 DNA polymerase and nicking enzyme, the exponential amplification was achieved, generating numerous G-rich sequences. By the addition of K+ , G-rich sequences folded into G-quadruplex structures, which selectively interacted with NMM, obtaining an enhanced fluorescent signal. While Dam MTase was not present, LSLP maintained the original and stable hairpin structure, and could not trigger SDA and ERCA. So, G-rich sequence was not generated and interacted with NMM, leading to a weak background fluorescent signal. The advantages of this method were following points: (1) In the LSLP, the trigger strand was fully sealed by the long stem, effectively avoiding background signal caused by leakage of probe; (2) The hairpin probe with overhang as a template was used for SDA, avoiding nonspecific amplification caused by the folding of linear template; (3) The ligation reaction relying on the produced primer effectively improved the specificity; (4) The circular probe, which contained three recognition sites of nicking enzyme and two complementary sequences of G-rich sequence, effectively increased the signal output; (5) The effective combination of SDA and ERCA ensured the highly sensitivity for Dam MTase activity assay.
3.2. Feasibility of the Dam MTase activity assay To validate the feasibility of the sensing system, we monitored the fluorescence emission spectra at different conditions. As displayed in Fig. 1A, the system only with NMM exhibited extremely
weak fluorescent intensity (Fig. 1A, curve a). The fluorescent intensity was also week in the absence of hairpin probe or DpnI (Fig. 1A, curve b and c), suggesting the needing of DpnI and hairpin probe for the cleavage and amplification reactions. The system without Dam MTase showed a slightly enhanced fluorescent intensity (Fig. 1A, curve d). After Dam MTase was added, an enhanced fluorescent intensity was observed (Fig. 1A, curve e), indicating that the methylation and cleavage reactions were happened and the produced ssDNA initiated SDA and RCA. Compared with it, after both Dam MTase and enough Nt.BbvCI were added, the fluorescent intensity dramatically increased (Fig. 1A, curve f). The result indicated that the methylation and cleavage reactions were happened and the produced ssDNA initiated SDA and ERCA in the presence of Dam MTase and enough Nt.BbvCI. Additionally, PAGE was also utilized to demonstrate the feasibility of methylation and cleavage reactions (Fig. 1B). One bright band was obviously observed (Fig. 1B, line 1) in the absence of Dam MTase and DpnI, suggesting that the stable LSLP formed. And we also saw a similar band (Fig. 1B, line 2) in the presence of DpnI, indicating that DpnI could not cleave the unmethylated LSLP. However, when both Dam MTase and DpnI were presented, the bright band darkened and a new band with lower molecular weight appeared. The result indicated that the LSLP was methylated by Dam MTase and the methylated LSLP was cleaved by DpnI. All of the above results were consistent with that of fluorescence emission spectra.
3.3. Optimization of the LSLP design LSLP was first optimized to achieved a good analytical performance. In LSLP, the sequence length between the recognition site of Dam MTase and loop (defined as buffer zone) played an important role in reducing background signal and increasing the amplified signal. Six LSLPs with different sequence lengths of buffer zone were designed. Here, we investigated the effects of different lengths of buffer zone on F/F0 , where F was fluorescent intensity with Dam MTase. As shown in Fig. 2, the F/F0 increased with the increase of the sequence length of buffer zone from 1 to 3 base pairs (Probe 1, Probe 2 and Probe 3). The reason was that the longer buffer zone with more base pairs could protect the recognition site of Dam MTase to achieve effective enzymatic reaction. However, the F/F0 decreased with the further increase of the sequence length of buffer zone from 4 to 7 base pairs (Probe 4, Probe 5 and Probe 6). The result indicated that the new hairpin probe produced by the cleavage of DpnI was relatively stable and was difficult to undergo a conformational change to ssDNA for initiating subsequent amplification
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Fig. 1. (A) Fluorescence response of the Dam MTase activity assay system at different conditions: (a) NMM, (b) Dam MTase + DpnI + Nt.BbvCI (6 U) + NMM, (c) Dam MTase + hairpin probe + Nt.BbvCI (6 U) + NMM, (d) DpnI + hairpin probe + Nt.BbvCI (6 U) + NMM, (e) Dam MTase + DpnI + hairpin probe + Nt.BbvCI (2 U) + NMM, (f) Dam MTase + DpnI + hairpin probe + Nt.BbvCI (6 U) + NMM. (B) Nondenaturing polyacrylamide gel (15%) electrophoresis analysis of Dam MTase activity. Lane 1, LSLP (200 nM); Lane 2, LSLP (200 nM) and DpnI (4 U); Lane 3, LSLP (200 nM), DpnI (4 U) and Dam MTase (16 U/mL).
Fig. 2. The influences of different sequence lengths of the buffer zone in LSLP on F/F0 .
reactions, due to the high Tm value. Therefore, Probe 3 with 3 base pairs of buffer zone was selected as the optimal design.
depended on the ability of trigger strand to hybridize to template [37,38]. So, F/F0 increased with the increasing of low concentration of hairpin probe from 2 to 40 nM. And, high concentration of hairpin probe with overhang as the template used for SDA avoided nonspecific amplification caused by the folding of linear template [39]. So, F/F0 almost unchanged with the increasing of high concentration of hairpin probe from 40 to 60 nM. Finally, 40 nM hairpin probe and 3 U Phi29 DNA polymerase were used as the optimal conditions, respectively. Furthermore, the produced G-quadruplex structure interacted with NMM, which affected effective signal output. And the effect of NMM concentration on F/F0 was also studied (Fig. S4). When NMM concentration was 3 M, the maximum F/F0 was achieved. And regardless of whether NMM concentration increased or not, the F/F0 decreased. So, 3 M NMM was used in the subsequent studies. Besides, the assay was influenced by the reaction time, including methylation and cleavage (MC), SDA, ERCA and NMM intercalation reaction time. As shown in Fig. S5–S8, the optimal MC, SDA, ERCA and NMM intercalation reaction time were 2 h, 30 min, 3 h and 30 min, respectively.
3.4. Optimization of experimental conditions 3.5. Performance study of the amplified Dam MTase assay Taking into account the influences of experimental conditions on fluorescent intensity, several experimental parameters including hairpin probe, DpnI, Phi29 DNA polymerase, and NMM concentrations needed to be optimized to improve the performance of this method. DpnI cleaved the methylated LSLP, which was important for effective signal transduction. First, the effect of DpnI concentration on F/F0 was studied (Fig. S1). We clearly saw that the F/F0 was strongly dependent on the DpnI concentration. And F/F0 increased with the increasing of low concentration of DpnI from 0.2 to 2 U, while F/F0 almost unchanged with the increasing of high concentration of DpnI from 2 to 4 U. So, 2 U DpnI was the optimal concentration. Subsequently, hairpin probe and Phi29 DNA polymerase concentrations that affected effective signal amplification were optimized. Fig. S2-S3 showed the effects of these factors on F/F0 . When hairpin probe concentration and Phi29 DNA polymerase concentration were 40 nM and 3 U, respectively, the corresponding F/F0 reached the maximum platform. The possible reason was that low concentration of hairpin probe as the template limited the amplification efficiency because the amplification efficiency of SDA
Under the optimal experimental conditions, a series of samples containing different concentrations of Dam MTase were analyzed. Fig. 3A showed that fluorescent intensity was dependent on the Dam MTase concentration. The fluorescent intensity increased with the increasing concentration of Dam MTase from 0 to 20 U/mL. It was because that higher concentrations of Dam MTase effectively catalyzed methylation reaction, more G-quadruplex structures were produced after the amplification reactions, which interacted with NMM, resulting in significantly enhanced fluorescent intensity. While the rate of change in fluorescent intensity became small at high concentration of Dam MTase, suggesting that most of the LSLPs were methylated. And we used the net signal F (F = F–F0 ) for representing the changes of fluorescent signal in response to different concentrations of Dam MTase (Fig. 3B). Both F and Dam MTase concentration in a range of 4.0 × 10−4 to 1.0 × 10−2 U/mL, had a good linear relationship with correlation coefficient of 0.9989 (inset in Fig. 3B). Based on the principle of three times standard deviation, the detection limit was calculated to be 8.1 × 10−5 U/mL. The linear range was acceptable in comparison to previously reported methods [19,20,26,28,31,41–45]. The
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Fig. 3. (A) Fluorescence spectra of the system treated with different concentrations of Dam MTase. (B) The relationship between fluorescence enhancement and Dam MTase concentration. The inset shows the linear plot of F vs Dam MTase concentration. Table 1 Comparison of linear range and detection limit of different methods for DNA MTase activity detection. Method
Strategy
Linear range (U/mL)
Detection limit (U/mL)
Reference
Colorimetric Colorimetric Colorimetric
Enzyme-responsive Au nanoparticle assembly and disassembly DNA-modified gold nanoparticles coupled with enzyme-linkage reactions Unmodified Au nanorods as a signal sensing probe coupled with enzyme-linkage reactions Methylation-blocked cascade amplification strategy Hairpin fluorescent DNA probe coupled with enzyme-linked reaction Hairpin-shaped DNAzyme-based fluorescent amplification strategy Transcription-mediated duplex-specific nuclease-assisted cyclic signal amplification DNA-templated silver nanoclusters without rapid restriction enzyme allosteric molecular beacon Combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension Discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide microelectrode Electrocatalytic oxidation of ascorbic acid-based sensing platform Methylation-triggered autonomous exonuclease III-assisted isothermal cycling signal amplification Loop-mediated cascade amplification strategy based on SDA and ERCA
– 1.0–10 0.2–30
2.5 0.3 0.14
[40] [41] [42]
0.8–24 0.8–40 0.4–20 0.05–10 0.4–20 0.7–30 0.1–20
0.4 0.8 0.4 0.015 0.1 0.57 0.04
[43] [26] [31] [28] [44] [45] [19]
0.5–50
0.18
[20]
0.05–200 0.004–4.0
0.025 0.004
[46] [21]
0.0004–0.01
8.1 × 10−5
This work
Colorimetric Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Electrochemical Electrochemical Electrochemical Electrochemical Fluorescent
detection limit was about four orders of magnitude lower than that of the reported colorimetric [41–43] and fluorescent [26,31,44,45] methods, and about three orders of magnitude lower than that of the reported electrochemical method [19,46]. The details of the comparisons were listed in Table 1. And the improved results were mainly due to the several points: (1) the low background leakage of LSLP; (2) the improved specificity of SDA and ligation reaction and (3) the high amplification efficiency of SDA and ERCA. Moreover, four DNA MTases were selected to evaluate the specificity of the assay, including AluI (5 -AGCT-3 ), HhaI (5 -GCGC-3 ), M.SssI (5 -CCGG-3 ) and HaeIII (5 -GGCC-3 ) MTases (Fig. 4). An obvious fluorescent intensity was observed by the addition of 8 U/mL Dam MTase. However, the negligible fluorescent intensities produced by the addition of 8 U/mL other MTases were almost the same as the low background intensity. The above data showed that this method had a high specificity for the detection of Dam MTase activity. Next, the precision and repeatability of the assay were studied. And the relative standard deviations (RSD) were calculated for the assessment of precision and repeatability. The RSD (n = 3) obtained from the same batch were 3.2%, 2.8% and 1.2% at the Dam MTase concentrations of 8.0 × 10−4 U/mL, 2.0 × 10−3 U/mL and 8.0 × 10−3 U/mL, respectively. The RSD of batch-to-batch measurements were 4.6%, 2.8% and 2.2% at same Dam MTase concentrations in three days. The above analysis data showed that the method had a high precision and repeatability for the detection of Dam MTase activity. Furthermore, the applicability of the assay in complex biological samples were studied. Different concentrations of Dam MTase
Fig. 4. The selectivity of the assay for Dam MTase and other MTases including AluI, HaeIII, M.SssI, and HhaI MTases. The concentrations of the all DNA MTases are 8 U/mL.
(8.0 × 10−4 , 2.0 × 10−3 and 8.0 × 10−3 U/mL) were spiked in 10% diluted human serum samples, obtaining the corresponding recoveries (Table S2). The obtained recoveries were from 95% to 98.7% with the corresponding RSD of 4.1%, 3.0% and 2.6%, respectively. The above results demonstrated that our Dam MTase activity assay had a potential for MTase detection in complex biological samples.
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Fig. 5. (A) The inhibition effect of 1 M different inhibitors on Dam MTase activity. Three inhibitors: gentamycin, benzylpenicillin and 5–fluorouracil. (B) The inhibition effect of different concentrations of 5–fluorouracil on Dam MTase activity.
3.6. Dam MTase activity inhibition assay
Acknowledgements
As far as we know, Dam MTase activity inhibitors were considered to be potential anticancer agents. Here, three inhibitors containing gentamycin, benzylpenicillin and 5–fluorouracil were selected to investigate whether the method could be used for inhibition evaluation and inhibitor screening. Taking into account a variety of auxiliary enzymes involved in our experiment, the influences of the three inhibitors on auxiliary enzymes were firstly investigated (Fig. S12). It was found that the corresponding fluorescent intensities were not significant changed, compared with the fluorescent intensity obtained from only the addition of Dam MTase. These results demonstrated that three inhibitors did not have any impact on auxiliary enzymes activity, when the inhibitors concentrations were not more than 1 M. Then, we studied the effects of 1 M three inhibitors on the Dam MTase activity (Fig. 5A). And by comparing relative activity of Dam MTase with adding different inhibitors, we could clearly see that 5–fluorouracil achieved better inhibition effect on Dam MTase activity at same concentration, due to its higher toxicity. Subsequently, the influences of different concentrations of 5–fluorouracil on Dam MTase activity were also studied. Fig. 5B showed that relative activity of Dam MTase decreased with the increasing concentration of 5–fluorouracil, demonstrating that the inhibition effect of Dam MTase activity was dose-dependent. The half-maximal inhibitory concentration (IC50) value of 5–fluorouracil was estimated to be 0.8 M. All the above results demonstrate the potential use of the DNA MTase activity assay method for inhibition evaluation and inhibitor screening.
This work was supported by the National Natural Science Foundation of China (Grant nos. 21375078, 21475077, 21675100 and 21675101).
4. Conclusions In conclusion, a loop-mediated cascade amplification fluorescent method was explored for DNA MTase activity detection based on SDA and ERCA. The trigger strand for subsequent amplification was fully sealed by the long stem of LSLP, effectively avoiding the nonspecific amplification caused by the leakage of probe and ensuring the accurate detection. And the highly sensitivity was achieved by the effective combination of SDA and ERCA. Due to the above advantages, this method could detect as low as 8.1 × 10−5 U/mL DNA MTase, which was superior or comparable to that of the reported literature. And this method could well distinguish Dam MTase from other MTases. Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase using antibiotics and anti-cancer drug as model. These results demonstrate that our system can be used as a potential biological analysis tool for basic research and early cancer diagnosis.
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Biographies Wanling Cui is currently pursuing her Ph.D. degree in Shandong University. Her research interests focuses on construction of biosensor for biological analysis. Lei Wang received her Ph.D. degree in Shandong University, and now is a professor in Shandong University. Her research interests focuses on development of DNA nanomaterial based new strategies for drug analysis, and DNA nanomaterial based diagnosis and therapy of cancer. Xiaowen Xu received his Ph.D. degree from Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. And he is research assistant in Shandong University. His current research interests focuses on DNA/nanoparticles system based biological analysis and construction of logic gate. Yan Wang is a Associate Chief Physician and now is in the 88th Hospital of PLA of general surgery. Wei Jiang received his Ph.D. degree from Shandong University and now is a professor in Shandong University. His research interests focuses on application of DNA nanomaterials in biological analysis and development of signal amplification strategy for biological molecules detection.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A loop-mediated cascade amplification strategy for highly sensitive detection of DNA methyltransferase activity Wanling Cui a , Lei Wang b , Xiaowen Xu a , Yan Wang c,∗∗ , Wei Jiang a,∗ a Key Laboratory for Colloid and Interface Chemistry of Education Ministry, School of Chemistry and Chemical Engineering, Shandong University, 250100 Jinan, P.R. China b School of Pharmaceutical Sciences, Shandong University, 250012 Jinan, P.R. China c The 88th Hospital of PLA, 270100 Tai’an, P.R. China
a r t i c l e
i n f o
Article history: Received 30 August 2016 Received in revised form 6 December 2016 Accepted 3 January 2017 Available online 8 January 2017 Keywords: DNA methyltransferase activity Long stem-loop probe Strand displacement amplification Exponential rolling circle amplification Fluorescent detection
a b s t r a c t DNA methyltransferase (MTase) is a predictive cancer biomarker and drug target, and the sensitive detection of DNA MTase activity is crucial to early cancer diagnosis and therapy. In this work, we developed a loop-mediated cascade amplification strategy for highly sensitive fluorescent detection of DNA MTase activity based on strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Firstly, we designed a long stem-loop probe (LSLP) which contains a methylation site for DNA MTase recognition, a long stem for ensuring the stability of probe, and a loop for initiating subsequent amplification. The loop and part of stem of LSLP acted as a trigger strand for subsequent signal output process. And the trigger strand was fully enclosed in loop of LSLP by the long stem, avoiding the nonspecific amplification caused by the leakage of probe. The LSLP was methylated by DNA MTase and then was specifically cleaved by DpnI endonuclease, producing a trigger strand. Under the synergetic action of polymerase and nicking enzyme, the trigger strand initiated SDA, producing many primers. The produced primers initiated ERCA, synthesizing numerous G-quadruplex sequences. The G-quadruplex sequences interacted with N-methylmesoporphyrin IX, obtaining an enhanced fluorescent signal. The method could detect as low as 8.1 × 10−5 U/mL DNA MTase. Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase activity. These results show that our system has a great potential in early cancer diagnosis and therapy. © 2017 Elsevier B.V. All rights reserved.
1. Introduction DNA methyltransferase (MTase) is an epigenetic modification enzyme, which plays a vital role in many important biological processes, including the regulation of gene expression, developmental regulation and genomic imprinting [1–3]. It catalyzes DNA methylation by the covalent addition of methyl groups to adenine or cytosine in the corresponding recognition sequences [4–7]. Studies have shown that abnormal DNA MTase activity is closely related to the initiation and progression of diseases such as cancer [8–10]. Accordingly, DNA MTase activity has been widely seen as a predictive cancer biomarker and drug target [11–14]. Therefore, the sensitive detection of DNA MTase activity is of significant importance for DNA MTase related cancer diagnosis and therapy.
∗ Corresponding author. ∗∗ Corresponding author. E-mail addresses: [email protected] (Y. Wang), [email protected] (W. Jiang). http://dx.doi.org/10.1016/j.snb.2017.01.013 0925-4005/© 2017 Elsevier B.V. All rights reserved.
Traditionally, radioactive labeling [15], gel electrophoresis [16], and high-performance liquid chromatography [17,18] are used for the detection of DNA MTase activity. In addition to the above methods, many new strategies for the detection of DNA MTase activity have been developed to improve the detection sensitivity and specificity, including electrochemical [19–21], chemiluminescent [22,23], colorimetric [24,25] and fluorescent methods [26–29]. Among them, fluorescent methods as powerful bioanalytical tools for DNA MTase activity assay have received the widespread attention. In general, fluorescent methods have been established by using double-stranded DNA (dsDNA) probe or hairpin probe with overhang to achieve target recognition and signal transduction [30–34]. The signal transduction strand is partially enclosed in the dsDNA probe or stem of hairpin probe. Under the recognition action of DNA MTase, the dsDNA probe or hairpin probe releases a signal transduction strand and then the signal transduction strand triggers the subsequent signal output process. The partially enclosed manner leads to nonspecific amplification caused by leakage of probe, resulting in false positive signal. However, the competing
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hybridization between dsDNA probe or hairpin probe and report probe happens in the absence of DNA MTase [35,36], which may causing the nonspecific background amplification, compromising detection sensitivity and accuracy. In order to solve those problems, we developed a loop-mediated cascade amplification strategy for highly sensitive fluorescent detection of DNA MTase activity based on strand displacement amplification (SDA) and exponential rolling circle amplification (ERCA). Firstly, we designed a long stem-loop probe (LSLP) which contains a methylation site for DNA adenine methylation (Dam) MTase recognition, a long stem for ensuring the stability of probe, and a loop for initiating subsequent amplification. The loop and part of stem of LSLP acted as a trigger strand for subsequent signal output process. And the trigger strand was fully enclosed in loop of LSLP by the long stem, avoiding the nonspecific amplification caused by the leakage of probe. The LSLP was methylated by DNA MTase and then was specifically cleaved by DpnI endonuclease (DpnI), producing a trigger strand. Then, the trigger strand initiated SDA and ERCA, synthesizing large numbers of G-rich sequences. Finally, the G-rich sequences selectively interacted with N-methylmesoporphyrin IX (NMM), obtaining an enhanced fluorescent signal. By effective combination of LSLP design and cascade amplification strategy, this method could detect as low as 8.1 × 10−5 U/mL DNA MTase, which is superior or comparable to that of the reported literature. Moreover, this method could well distinguish Dam MTase from other MTases (AluI, HhaI, M.SssI and HaeIII MTases). Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase activity using gentamycin, benzylpenicillin and 5–fluorouracil as model. These results shows that our system has a great potential in early cancer diagnosis and therapy. 2. Experimental section 2.1. Materials and apparatus Synthetic DNA oligonucleotides were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China), and their sequences were shown in Table S1 in the Supporting Information. DNA adenine methylation (Dam), HaeIII, HhaI, M.SssI, and AluI MTase, DpnI endonuclease, S-adenosyl-l-methionine (SAM), Klenow Fragment (3 –5 exo-) polymerase, T4 DNA ligase, and Nt.BbvCI were obtained from New England Biolabs (Ipswich, MA, USA). Gentamycin, benzylpenicillin, 5–fluorouracil were purchased from Sangon Biotechnology Co., Ltd. (Shanghai, China) and used as received. Phi29 DNA polymerase was from Fermentas (Lithuania). All other reagents used in this work were analytical grade and used without further purification or modification. All solutions were prepared in ultrapure water (> 18.25 M cm) from a Millipore Milli-Q water purification system. The fluorescence spectra were recorded using Hitachi F-7000 fluorescence spectrometer (Hitachi. Ltd., Japan). The emission spectra were collected from 560 to 690 nm and excitation wavelength was set at 399 nm. The fluorescence emission intensity was measured at 618 nm. Both excitation and emission slit widths were all set at 10.0 nm and the photomultiplier tube voltage was 700 V. 2.2. Assay of Dam MTase activity In the typical methylation and cleavage experiments, 20 nM LSLP, 160 M SAM, 2 U DpnI and various amounts of Dam MTase were put into 10 L of methylase buffer (50 mM NaCl, 10 mM TrisHCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.5) and incubated at 37 ◦ C for 2 h. Then, 40 nM hairpin probe, 1 U Klenow Fragment (3 –5 exo-), 0.6 mM dNTPs, 2 U Nt.BbvCI and 1 × CutSmart (50 mM KAc, 20 mM Tris-HAc, 10 mM Mg (Ac) 2 , 100 g/mL BSA, pH 7.9) were
added. After the incubation at 37 ◦ C for 30 min, the reaction mixture was heated at 85 ◦ C for 20 min to deactivate the enzymes. After that, 120 U T4 DNA ligase, 800 nM padlock probe and 1 × T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2 , 10 mM dithiothreitol, 1 mM ATP, pH 7.5) were put into the above reaction mixture and kept at 37 ◦ C for 1 h to obtain the circular probe. Subsequently, 1 mM dNTPs, 3 U Phi29 DNA polymerase, 4 U Nt.BbvCI and 1 × CutSmart were added. The reaction was carried out at 37 ◦ C for 3 h, and then terminated by heating to 75 ◦ C for 20 min. Finally, 200 M KCl and 3 M NMM were added and incubated at 37 ◦ C for 30 min, followed by the fluorescence measurement. 2.3. Gel electrophoresis to study methylation and cleavage reactions The gel electrophoresis was used to determine whether or not the methylation and cleavage reactions were carried out, by separating the DpnI-cleaved products from the LSLP. The different samples were analyzed by putting them on a 15% nondenaturating polyacrylamide gel electrophoresis (PAGE). The electrophoresis was performed in 1 × Tris-borate-EDTA (TBE) buffer (89 mM Tris, 89 mM Boric Acid, 2.0 mM EDTA, pH 8.3) at 30 mA for 1.5 h, followed by ethidium bromide staining. The gels were photographed under UV imaging system (Bio-RAD Laboratories Inc., USA). 2.4. Effect of inhibitors on Dam MTase activity In inhibition assay, we chose three inhibitors (gentamycin, benzylpenicillin, and 5–fluorouracil) to investigate the inhibition effect of inhibitors on Dam MTase activity. The inhibition experiment was performed in same procedures as that mentioned above for Dam MTase activity assay, except for needing add various concentrations of the inhibitors. The inhibition effect of inhibitors was evaluated by the relative activity of Dam MTase, which could be expressed by the equation: relative activity = (F2 –F0 )/(F1 –F0 ), where F2 and F1 were fluorescent intensity of 8 U/mL Dam MTase with or without inhibitor respectively, and F0 was fluorescent intensity without Dam MTase. Our assay system contained auxiliary enzymes (DpnI, Klenow Fragment (3 –5 exo-) polymerase, T4 DNA ligase, and Nt.BbvCI, and Phi29 DNA polymerase). Control experiments were used to investigate whether or not inhibitors had effect on the activities of those enzymes. The methylation experiment was carried out at 37 ◦ C for 3 h to obtain the absolutely methylated probe, and then was heated at 65 ◦ C for 20 min to terminate the reaction. Subsequently, 1 M inhibitor and the other reagents were added to investigate inhibition effect of inhibitor on auxiliary enzymes. 3. Results and discussion 3.1. Principle of cascade amplification strategy for Dam MTase activity assay Scheme 1 outlined the design principle of sensing system for DNA MTase activity assay. Due to the same recognition site of Dam MTase and DpnI, they were respectively selected as the model DNA MTase and methylation-sensitive restriction endonuclease to verify the feasibility of the method. In the presence of Dam MTase, Dam MTase recognized and methylated the LSLP to form the methylated LSLP containing the sequences of 5 -GmATC-3 . Then, DpnI specifically cleaved the methylated LSLP between the two methyladenines into two parts. One part was a dsDNA, and the other one was a new hairpin probe. Under the experimental conditions, the new hairpin probe was unstable and underwent a conformational change to single-stranded DNA (ssDNA), due to the low melting temperature (Tm ) value. The ssDNA hybridized with the overhang
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Scheme 1. Experimental principle of the loop-mediated cascade amplification strategy for DNA MTase activity detection through SDA and ERCA. In long stem-loop probe, the green segment is the recognition site of DNA MTase, the purple loop segment is the trigger sequence that can trigger SDA, and the blue stem segment is used to close the trigger sequence. In hairpin probe, the purple segment is complementary to the trigger sequence in long stem-loop probe, and the light blue segment is the recognition site of Nt.BbvCI nicking endonuclease. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
of hairpin probe and acted as a trigger strand to trigger a polymerization reaction in the presence of Klenow Fragment polymerase and dNTPs, yielding a duplex strand with a full recognition site of Nt.BbvCI nicking enzyme. After the cleavage of Nt.BbvCI nicking enzyme, a new replication site for next polymerization reaction was produced. Under the synergetic action of polymerase and nicking enzyme, such repeated polymerization and nicking reactions happened, releasing many primers. Every one of the released primers could hybridize with the padlock probe in the presence of T4 DNA ligase to obtain a circular probe, which contained three recognition sites of Nt.BbvCI, two complementary sequences of G-rich sequence and a binding region of primer. Subsequently, by the addition of Phi29 DNA polymerase, the primer triggered RCA, synthesizing a long DNA product containing many concatenated sequences. Then, the long DNA product hybridized with multiple padlock probes, forming stable dsDNAs with multiple recognition sites of Nt.BbvCI. Next, Nt.BbvCI cleaved the stable dsDNAs into short dsDNAs. Due to the low melting temperature value of short dsDNAs, large numbers of G-rich sequences and primers that used for next RCA were produced. Through the synergetic action of Phi29 DNA polymerase and nicking enzyme, the exponential amplification was achieved, generating numerous G-rich sequences. By the addition of K+ , G-rich sequences folded into G-quadruplex structures, which selectively interacted with NMM, obtaining an enhanced fluorescent signal. While Dam MTase was not present, LSLP maintained the original and stable hairpin structure, and could not trigger SDA and ERCA. So, G-rich sequence was not generated and interacted with NMM, leading to a weak background fluorescent signal. The advantages of this method were following points: (1) In the LSLP, the trigger strand was fully sealed by the long stem, effectively avoiding background signal caused by leakage of probe; (2) The hairpin probe with overhang as a template was used for SDA, avoiding nonspecific amplification caused by the folding of linear template; (3) The ligation reaction relying on the produced primer effectively improved the specificity; (4) The circular probe, which contained three recognition sites of nicking enzyme and two complementary sequences of G-rich sequence, effectively increased the signal output; (5) The effective combination of SDA and ERCA ensured the highly sensitivity for Dam MTase activity assay.
3.2. Feasibility of the Dam MTase activity assay To validate the feasibility of the sensing system, we monitored the fluorescence emission spectra at different conditions. As displayed in Fig. 1A, the system only with NMM exhibited extremely
weak fluorescent intensity (Fig. 1A, curve a). The fluorescent intensity was also week in the absence of hairpin probe or DpnI (Fig. 1A, curve b and c), suggesting the needing of DpnI and hairpin probe for the cleavage and amplification reactions. The system without Dam MTase showed a slightly enhanced fluorescent intensity (Fig. 1A, curve d). After Dam MTase was added, an enhanced fluorescent intensity was observed (Fig. 1A, curve e), indicating that the methylation and cleavage reactions were happened and the produced ssDNA initiated SDA and RCA. Compared with it, after both Dam MTase and enough Nt.BbvCI were added, the fluorescent intensity dramatically increased (Fig. 1A, curve f). The result indicated that the methylation and cleavage reactions were happened and the produced ssDNA initiated SDA and ERCA in the presence of Dam MTase and enough Nt.BbvCI. Additionally, PAGE was also utilized to demonstrate the feasibility of methylation and cleavage reactions (Fig. 1B). One bright band was obviously observed (Fig. 1B, line 1) in the absence of Dam MTase and DpnI, suggesting that the stable LSLP formed. And we also saw a similar band (Fig. 1B, line 2) in the presence of DpnI, indicating that DpnI could not cleave the unmethylated LSLP. However, when both Dam MTase and DpnI were presented, the bright band darkened and a new band with lower molecular weight appeared. The result indicated that the LSLP was methylated by Dam MTase and the methylated LSLP was cleaved by DpnI. All of the above results were consistent with that of fluorescence emission spectra.
3.3. Optimization of the LSLP design LSLP was first optimized to achieved a good analytical performance. In LSLP, the sequence length between the recognition site of Dam MTase and loop (defined as buffer zone) played an important role in reducing background signal and increasing the amplified signal. Six LSLPs with different sequence lengths of buffer zone were designed. Here, we investigated the effects of different lengths of buffer zone on F/F0 , where F was fluorescent intensity with Dam MTase. As shown in Fig. 2, the F/F0 increased with the increase of the sequence length of buffer zone from 1 to 3 base pairs (Probe 1, Probe 2 and Probe 3). The reason was that the longer buffer zone with more base pairs could protect the recognition site of Dam MTase to achieve effective enzymatic reaction. However, the F/F0 decreased with the further increase of the sequence length of buffer zone from 4 to 7 base pairs (Probe 4, Probe 5 and Probe 6). The result indicated that the new hairpin probe produced by the cleavage of DpnI was relatively stable and was difficult to undergo a conformational change to ssDNA for initiating subsequent amplification
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Fig. 1. (A) Fluorescence response of the Dam MTase activity assay system at different conditions: (a) NMM, (b) Dam MTase + DpnI + Nt.BbvCI (6 U) + NMM, (c) Dam MTase + hairpin probe + Nt.BbvCI (6 U) + NMM, (d) DpnI + hairpin probe + Nt.BbvCI (6 U) + NMM, (e) Dam MTase + DpnI + hairpin probe + Nt.BbvCI (2 U) + NMM, (f) Dam MTase + DpnI + hairpin probe + Nt.BbvCI (6 U) + NMM. (B) Nondenaturing polyacrylamide gel (15%) electrophoresis analysis of Dam MTase activity. Lane 1, LSLP (200 nM); Lane 2, LSLP (200 nM) and DpnI (4 U); Lane 3, LSLP (200 nM), DpnI (4 U) and Dam MTase (16 U/mL).
Fig. 2. The influences of different sequence lengths of the buffer zone in LSLP on F/F0 .
reactions, due to the high Tm value. Therefore, Probe 3 with 3 base pairs of buffer zone was selected as the optimal design.
depended on the ability of trigger strand to hybridize to template [37,38]. So, F/F0 increased with the increasing of low concentration of hairpin probe from 2 to 40 nM. And, high concentration of hairpin probe with overhang as the template used for SDA avoided nonspecific amplification caused by the folding of linear template [39]. So, F/F0 almost unchanged with the increasing of high concentration of hairpin probe from 40 to 60 nM. Finally, 40 nM hairpin probe and 3 U Phi29 DNA polymerase were used as the optimal conditions, respectively. Furthermore, the produced G-quadruplex structure interacted with NMM, which affected effective signal output. And the effect of NMM concentration on F/F0 was also studied (Fig. S4). When NMM concentration was 3 M, the maximum F/F0 was achieved. And regardless of whether NMM concentration increased or not, the F/F0 decreased. So, 3 M NMM was used in the subsequent studies. Besides, the assay was influenced by the reaction time, including methylation and cleavage (MC), SDA, ERCA and NMM intercalation reaction time. As shown in Fig. S5–S8, the optimal MC, SDA, ERCA and NMM intercalation reaction time were 2 h, 30 min, 3 h and 30 min, respectively.
3.4. Optimization of experimental conditions 3.5. Performance study of the amplified Dam MTase assay Taking into account the influences of experimental conditions on fluorescent intensity, several experimental parameters including hairpin probe, DpnI, Phi29 DNA polymerase, and NMM concentrations needed to be optimized to improve the performance of this method. DpnI cleaved the methylated LSLP, which was important for effective signal transduction. First, the effect of DpnI concentration on F/F0 was studied (Fig. S1). We clearly saw that the F/F0 was strongly dependent on the DpnI concentration. And F/F0 increased with the increasing of low concentration of DpnI from 0.2 to 2 U, while F/F0 almost unchanged with the increasing of high concentration of DpnI from 2 to 4 U. So, 2 U DpnI was the optimal concentration. Subsequently, hairpin probe and Phi29 DNA polymerase concentrations that affected effective signal amplification were optimized. Fig. S2-S3 showed the effects of these factors on F/F0 . When hairpin probe concentration and Phi29 DNA polymerase concentration were 40 nM and 3 U, respectively, the corresponding F/F0 reached the maximum platform. The possible reason was that low concentration of hairpin probe as the template limited the amplification efficiency because the amplification efficiency of SDA
Under the optimal experimental conditions, a series of samples containing different concentrations of Dam MTase were analyzed. Fig. 3A showed that fluorescent intensity was dependent on the Dam MTase concentration. The fluorescent intensity increased with the increasing concentration of Dam MTase from 0 to 20 U/mL. It was because that higher concentrations of Dam MTase effectively catalyzed methylation reaction, more G-quadruplex structures were produced after the amplification reactions, which interacted with NMM, resulting in significantly enhanced fluorescent intensity. While the rate of change in fluorescent intensity became small at high concentration of Dam MTase, suggesting that most of the LSLPs were methylated. And we used the net signal F (F = F–F0 ) for representing the changes of fluorescent signal in response to different concentrations of Dam MTase (Fig. 3B). Both F and Dam MTase concentration in a range of 4.0 × 10−4 to 1.0 × 10−2 U/mL, had a good linear relationship with correlation coefficient of 0.9989 (inset in Fig. 3B). Based on the principle of three times standard deviation, the detection limit was calculated to be 8.1 × 10−5 U/mL. The linear range was acceptable in comparison to previously reported methods [19,20,26,28,31,41–45]. The
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Fig. 3. (A) Fluorescence spectra of the system treated with different concentrations of Dam MTase. (B) The relationship between fluorescence enhancement and Dam MTase concentration. The inset shows the linear plot of F vs Dam MTase concentration. Table 1 Comparison of linear range and detection limit of different methods for DNA MTase activity detection. Method
Strategy
Linear range (U/mL)
Detection limit (U/mL)
Reference
Colorimetric Colorimetric Colorimetric
Enzyme-responsive Au nanoparticle assembly and disassembly DNA-modified gold nanoparticles coupled with enzyme-linkage reactions Unmodified Au nanorods as a signal sensing probe coupled with enzyme-linkage reactions Methylation-blocked cascade amplification strategy Hairpin fluorescent DNA probe coupled with enzyme-linked reaction Hairpin-shaped DNAzyme-based fluorescent amplification strategy Transcription-mediated duplex-specific nuclease-assisted cyclic signal amplification DNA-templated silver nanoclusters without rapid restriction enzyme allosteric molecular beacon Combining DNA methylation-sensitive cleavage and terminal transferase-mediated extension Discrimination of the aggregation of long and short DNA on a negatively charged indium tin oxide microelectrode Electrocatalytic oxidation of ascorbic acid-based sensing platform Methylation-triggered autonomous exonuclease III-assisted isothermal cycling signal amplification Loop-mediated cascade amplification strategy based on SDA and ERCA
– 1.0–10 0.2–30
2.5 0.3 0.14
[40] [41] [42]
0.8–24 0.8–40 0.4–20 0.05–10 0.4–20 0.7–30 0.1–20
0.4 0.8 0.4 0.015 0.1 0.57 0.04
[43] [26] [31] [28] [44] [45] [19]
0.5–50
0.18
[20]
0.05–200 0.004–4.0
0.025 0.004
[46] [21]
0.0004–0.01
8.1 × 10−5
This work
Colorimetric Fluorescent Fluorescent Fluorescent Fluorescent Fluorescent Electrochemical Electrochemical Electrochemical Electrochemical Fluorescent
detection limit was about four orders of magnitude lower than that of the reported colorimetric [41–43] and fluorescent [26,31,44,45] methods, and about three orders of magnitude lower than that of the reported electrochemical method [19,46]. The details of the comparisons were listed in Table 1. And the improved results were mainly due to the several points: (1) the low background leakage of LSLP; (2) the improved specificity of SDA and ligation reaction and (3) the high amplification efficiency of SDA and ERCA. Moreover, four DNA MTases were selected to evaluate the specificity of the assay, including AluI (5 -AGCT-3 ), HhaI (5 -GCGC-3 ), M.SssI (5 -CCGG-3 ) and HaeIII (5 -GGCC-3 ) MTases (Fig. 4). An obvious fluorescent intensity was observed by the addition of 8 U/mL Dam MTase. However, the negligible fluorescent intensities produced by the addition of 8 U/mL other MTases were almost the same as the low background intensity. The above data showed that this method had a high specificity for the detection of Dam MTase activity. Next, the precision and repeatability of the assay were studied. And the relative standard deviations (RSD) were calculated for the assessment of precision and repeatability. The RSD (n = 3) obtained from the same batch were 3.2%, 2.8% and 1.2% at the Dam MTase concentrations of 8.0 × 10−4 U/mL, 2.0 × 10−3 U/mL and 8.0 × 10−3 U/mL, respectively. The RSD of batch-to-batch measurements were 4.6%, 2.8% and 2.2% at same Dam MTase concentrations in three days. The above analysis data showed that the method had a high precision and repeatability for the detection of Dam MTase activity. Furthermore, the applicability of the assay in complex biological samples were studied. Different concentrations of Dam MTase
Fig. 4. The selectivity of the assay for Dam MTase and other MTases including AluI, HaeIII, M.SssI, and HhaI MTases. The concentrations of the all DNA MTases are 8 U/mL.
(8.0 × 10−4 , 2.0 × 10−3 and 8.0 × 10−3 U/mL) were spiked in 10% diluted human serum samples, obtaining the corresponding recoveries (Table S2). The obtained recoveries were from 95% to 98.7% with the corresponding RSD of 4.1%, 3.0% and 2.6%, respectively. The above results demonstrated that our Dam MTase activity assay had a potential for MTase detection in complex biological samples.
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Fig. 5. (A) The inhibition effect of 1 M different inhibitors on Dam MTase activity. Three inhibitors: gentamycin, benzylpenicillin and 5–fluorouracil. (B) The inhibition effect of different concentrations of 5–fluorouracil on Dam MTase activity.
3.6. Dam MTase activity inhibition assay
Acknowledgements
As far as we know, Dam MTase activity inhibitors were considered to be potential anticancer agents. Here, three inhibitors containing gentamycin, benzylpenicillin and 5–fluorouracil were selected to investigate whether the method could be used for inhibition evaluation and inhibitor screening. Taking into account a variety of auxiliary enzymes involved in our experiment, the influences of the three inhibitors on auxiliary enzymes were firstly investigated (Fig. S12). It was found that the corresponding fluorescent intensities were not significant changed, compared with the fluorescent intensity obtained from only the addition of Dam MTase. These results demonstrated that three inhibitors did not have any impact on auxiliary enzymes activity, when the inhibitors concentrations were not more than 1 M. Then, we studied the effects of 1 M three inhibitors on the Dam MTase activity (Fig. 5A). And by comparing relative activity of Dam MTase with adding different inhibitors, we could clearly see that 5–fluorouracil achieved better inhibition effect on Dam MTase activity at same concentration, due to its higher toxicity. Subsequently, the influences of different concentrations of 5–fluorouracil on Dam MTase activity were also studied. Fig. 5B showed that relative activity of Dam MTase decreased with the increasing concentration of 5–fluorouracil, demonstrating that the inhibition effect of Dam MTase activity was dose-dependent. The half-maximal inhibitory concentration (IC50) value of 5–fluorouracil was estimated to be 0.8 M. All the above results demonstrate the potential use of the DNA MTase activity assay method for inhibition evaluation and inhibitor screening.
This work was supported by the National Natural Science Foundation of China (Grant nos. 21375078, 21475077, 21675100 and 21675101).
4. Conclusions In conclusion, a loop-mediated cascade amplification fluorescent method was explored for DNA MTase activity detection based on SDA and ERCA. The trigger strand for subsequent amplification was fully sealed by the long stem of LSLP, effectively avoiding the nonspecific amplification caused by the leakage of probe and ensuring the accurate detection. And the highly sensitivity was achieved by the effective combination of SDA and ERCA. Due to the above advantages, this method could detect as low as 8.1 × 10−5 U/mL DNA MTase, which was superior or comparable to that of the reported literature. And this method could well distinguish Dam MTase from other MTases. Furthermore, this assay was successfully used to assess the inhibition effect of inhibitors for DNA MTase using antibiotics and anti-cancer drug as model. These results demonstrate that our system can be used as a potential biological analysis tool for basic research and early cancer diagnosis.
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Biographies Wanling Cui is currently pursuing her Ph.D. degree in Shandong University. Her research interests focuses on construction of biosensor for biological analysis. Lei Wang received her Ph.D. degree in Shandong University, and now is a professor in Shandong University. Her research interests focuses on development of DNA nanomaterial based new strategies for drug analysis, and DNA nanomaterial based diagnosis and therapy of cancer. Xiaowen Xu received his Ph.D. degree from Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. And he is research assistant in Shandong University. His current research interests focuses on DNA/nanoparticles system based biological analysis and construction of logic gate. Yan Wang is a Associate Chief Physician and now is in the 88th Hospital of PLA of general surgery. Wei Jiang received his Ph.D. degree from Shandong University and now is a professor in Shandong University. His research interests focuses on application of DNA nanomaterials in biological analysis and development of signal amplification strategy for biological molecules detection.