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Enzyme-free and label-free signal amplification for monitoring endonuclease activity and inhibition via hybridization chain reaction† Jing Zhang, Zhilu Shi and Yan Jin* A label-free and enzyme-free amplification protocol has been proposed for studying endonuclease activity and inhibition on the basis of the enzyme-digested product triggered hybridization chain reaction (HCR). Three hairpin oligonucleotides were designed as probes which could not open or hybridize with each other at room temperature until the initiator DNA was released by specific enzymatic cleavage in the presence of endonuclease to trigger the hybridization chain reaction. SYBR Green I was chosen as a signal probe which intercalated into the grooves of the nicked double DNA polymer, generating a substantially apparent increase in fluorescence intensity. Once the activity of endonuclease is inhibited by enzyme inhibitors, the efficiency of HCR will be greatly decreased. Therefore, screening of endonuclease inhibitors can be achieved effectively as well as the assay of endonuclease activity. Meanwhile, the assay of endonuclease activity and inhibition achieves a better performance as compared to the previous
Received 13th February 2015, Accepted 22nd March 2015
reports. Importantly, it is a more universal method that can be simply used to study activity and inhibition
DOI: 10.1039/c5an00304k
of other endonucleases by changing the specific recognition site. So, the protocol was proved to be a sensitive and cost-effective approach for studying endonuclease activity and inhibition, and as such, it is
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promising for broad potential application in various biological reactions.
Introduction As a promising amplification technique for ultrasensitive bioanalysis, the hybridization chain reaction has aroused significant attention. HCR is based on the chain reaction of recognition and hybridization events between two hairpin DNA probes, which offer an enzyme-free amplification protocol for rapid detection of specific DNA sequences.1–4 HCR events can be triggered only in the presence of the initiator, and each copy of the initiator can trigger a cascade hybridization event.5–9 In addition, the most attractive advantage of HCR is that it allows for selective and specific extension at room temperature without enzyme-aided amplification. Based on the above advantages, numerous studies utilizing the amplification capability of HCR have been developed for detection of DNA, protein, small molecules and metal ions etc.10–16 For example, Zhang’s group combined HCR with enzyme-amplification to develop a fluorescence method for DNA detection by
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China. E-mail: [email protected]; Fax: (+) 86-29-81530727 † Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c5an00304k
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using a biotin-labeled hairpin probe and magnetic beads.17 Tang’s group reported an electrochemical immuno-HCR assay for determination of human IgG with DNA-functionalized gold nanoparticles and antibody-immobilized magnetic beads.18 Tan’s group put forward a fluorescence anisotropy strategy for ATP detection by combined target-triggered HCR with biotinmodified probes.19 Tang’s group constructed an electrochemical method for detection of Pb2+ based on the HCR reaction coupling with Pb2+-specific DNAzyme modified magnetic beads.13 As described above, HCR, by its unique assembly approach, has received widespread attention and application. However, to our knowledge, study on the biological reaction by HCR is rare. Herein, to explore further the application of the HCR technique for bioanalysis, a label-free fluorescence method had been developed for sensitive and accurate assay of endonuclease activity and inhibition by using SYBR Green I (SGI) as a signal probe. Protein–DNA interactions play important roles in life processes, including DNA replication, transcription, recombination and repair. The restriction endonuclease is a well-studied class of DNA-binding proteins and has been an important tool in the development of modern molecular biology. Therefore, the specific cleavage of DNA by restriction endonuclease is chosen as a model to explore the feasibility of studying biological reactions by HCR. In this case, three
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hairpin DNAs were designed and named as H1, H2 and H3 respectively. The initiator of HCR and the recognition site of EcoRI endonucleases are, respectively, embedded in the loop and stem part of H1. Thus, initiator strand can be specifically released when H1 was specifically cleaved by EcoRI endonucleases. Then, the initiator strand propagates a chain reaction of alternating H2 and H3 hybridization to form a nicked double-stranded polymer, leading to a significant increase in the fluorescence intensity. Once the activity of the enzyme was reduced by an inhibitor, the HCR efficiency was greatly affected. Therefore, it can be used to screen the endonuclease inhibitor. The assay of activity and inhibition of other endonucleases demonstrated that it offered a simple, rapid and sensitive platform to study biological process by HCR, which is of great theoretical and practical importance in bioanalysis and drug screening.
Experimental Chemicals EcoRI, BamHI endonucleases, T4 polynucleotide kinase (10 U µL−1) and T4 DNA ligase (5 U µL−1) were purchased from Sangon Biotech Co. (Shanghai, China). Lambda exonuclease (5 U µL−1) was purchased from New England Biolabs (NEB, U.K.). Lysozyme, immunoglobin G (IgG) and SYBR Green I (SGI, 19.7 mM) were purchased from Beijing Dingguo Biotechnology Co., Ltd (Beijing, China). DNA oligonucleotides used in this work are listed as follows: H1 (5′-CGCGCTGCGAATTCAGTCTAGGATTCGGCGTGGGTTAAGAATTCGCAGCGCG-3′), H2 (5′CACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG-3′), H3 (5′-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACT -3′), H*1 (5′-CGCGCTGCGGATCCAGTCTAGGATTCGGCGTGGGTTAAGGATCCGCAGCGCG-3′). They were synthesized by Shanghai Sangon Biotechnology Co. (Shanghai, China) and were all purified by reverse-phase highperformance liquid chromatography (HPLC). In the hairpin sequences, loops are italicized and sticky ends are underlined. The H1 oligonucleotide stock solutions were prepared with EcoRI buffer (50 mM Tris-HCl solution pH 7.5, 100 mM NaCl, 10 mM MgCl2). H2 and H3 DNA stock solutions were prepared in buffer: 0.4 M NaCl, 50 mM Na2HPO4 ( pH 7.5). Millipore MilliQ (18 MΩ cm) water was used in all experiments.
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Gel electrophoresis H1, H2 and H3 were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h before use. A 12.5% native polyacrylamide gel was prepared by using 1× TBE buffer (100 mM Tris-HCl, 83 mM boric acid, 1 mM EDTA, pH 8.0). The gel was run at 150 V for 40 min with a loading of 5 μL of each sample into the lanes at room temperature, stained with EB for 30 min. The visualization and photography were performed using a Molecular Imager with the Gel Doc system. Circular dichroism spectroscopy The CD spectra of the hairpin and double-stranded polymer were measured by using a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd, England, UK). CD spectra were recorded using a quartz cell of 1 mm optical path length and an instrument scanning speed of 100 nm min−1 with a response time of 2 s at room temperature. CD spectra were obtained by taking the average of three scans made from 210 to 320 nm. All the DNA samples were dissolved in the buffer (0.4 M NaCl, 50 mM Na2HPO4, pH 7.5) and heated to 90 °C for 5 min, gradually cooled to room temperature, and incubated at 4 °C overnight. The background of the buffer was subtracted from the CD data. Assay of EcoRI endonuclease activity and inhibition All the hairpin oligonucleotides were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h before use. 100 nM H1 was mixed with different concentrations of EcoRI endonuclease in 25 µL of EcoRI buffer (50 mM Tris-HCl solution 100 mM NaCl, 10 mM MgCl2, pH 7.5) and incubated at 37 °C for 1 h. After that, the above solution was added into 500 nM H2 and 500 nM H3 in 75 µL of reaction buffer (0.4 M NaCl, 50 mM Na2HPO4, pH 7.5). The total volume of the reaction solution was 100 µL. During this process, the hybridization chain reaction was triggered and progressed to form the long nicked DNA polymers. The inhibitor PP and 5-fluorouracil of EcoRI was incubated with 100 nM H1 in Tris-HCl buffer for 15 min at room temperature. Then, EcoRI was added and the resulting solution was incubated at 37 °C for 60 min. The detection procedure was the same as shown in the aforementioned assay of EcoRI endonuclease activity. All fluorescence measurements of the samples were carried out on a fluorometer F7000 (Hitachi, Japan) with excitation at 487 nm and an emission range from 505 to 600 nm, respectively.
Measurements All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Kyoto, Japan). The vertical electrophoresis system was purchased from Bio-Rad Laboratories, Inc. The Molecular Imager system was purchased from Shanghai Peiqing science & Technology. Co., Ltd (Shanghai, China). Circular Dichroism spectra were measured on a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd, England, UK).
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Results and discussion Sensing mechanism In this work, an enzyme-free method has been developed for assay of endonuclease activity and inhibition based on HCR amplification. As shown in Scheme 1, three hairpin probes were designed, including H1, H2 and H3. The stem of H1 contains the specific recognition site of EcoRI endonuclease
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Scheme 1 Schematic illustration of enzyme-free signal amplification for monitoring endonuclease activity and inhibition by the hybridization chain reaction.
(5′-d (GAATTC)-3′) and its loop can hybridize with the fragment I of H2. Both H2 and H3 have two fragments. The fragment I (blue line) at the 5′ end of H2 is complementary to the fragment I′ (blue line) of H3. The fragment II′ (red line) at the 3′ end of H3 can hybridize with the fragment II (red line) of H2. In general, H1, H2 and H3 could not open or hybridize with each other at room temperature in the absence of EcoRI endonucleases. Upon the addition of EcoRI endonucleases, H1 was specifically cleaved at the stem part and generated two fragments. According to the melting temperature (Tm) measurements, the loop fragment of H1 could not form a stable hairpin at room temperature. So, it acts as a single-stranded initiator to trigger the hybridization chain reaction. The initiator strand paired with the sticky end of H2, which undergoes a strand-displacement interaction to open the hairpin structure of H2. Then, the newly exposed sticky end of H2, fragment II, hybridized with the sticky end of H3 (fragment II′) and opened the hairpin of H3 to expose a sticky end (fragment I′) which is identical to the initiator strand in sequence and hybridized with the sticky end of another H2. In this way, each copy of the loop fragment of H1 can give rise to a chain reaction of hybridization events between alternating H2 and H3 hairpins to form a long double-helix. Once the activity of enzyme was restrained by enzyme inhibitors, the HCR efficiency was corresponding relatively reduced due to the low amount of HCR initiator. Therefore, the activity and inhibition of endonuclease can be sensitively monitored by analyzing the change in fluorescence intensity. Fluorescence analysis has been performed to verify the feasibility of the sensing scheme. As shown in Fig. 1A, the fluorescence of free SGI is weak. However, the fluorescence of SGI obviously increased from curve a to e when it was incubated with H2 and H3, because SGI can effectively bind to the double-stranded stem of hairpin probes. In the presence of EcoRI endonuclease, the fluorescence of SGI was enhanced significantly from curve e to f. That is, the specific cleavage of H1 by EcoRI-endonuclease-triggered HCR to form double-helix DNA polymers caused the intercalation of a large quantity of
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Fig. 1 Fluorescence spectra of SGI at different conditions. (A) (a) SGI, (b) H1 + SGI, (c) H1 + EcoRI + SGI, (d) H2 + H3 + SGI, (e) H1 + H2 + H3 + SGI, (f ) H1 + EcoRI + H2 + H3 + SGI. (B) (a) SGI, (b) H1 + SGI, (c) H1 + 5-fluorouracil + EcoRI + SGI, (d) H2 + H3 + SGI, (e) H1 + H2 + H3 + SGI, (f ) H1 + 5-fluorouracil + EcoRI + H2 + H3 + SGI.
SGI into the grooves of dsDNA. So, the EcoRI endonuclease activity can be studied by monitoring the variance of SGI fluorescence intensity. On the contrary, the fluorescence intensity increased slightly from the curve e to f in Fig. 1B when H1 was incubated with the inhibitors. That is, the HCR efficiency is greatly reduced once the activity of the EcoRI endonuclease is restrained in the presence of inhibitor. So, the preliminary results demonstrated that this label-free amplification strategy is feasible for study of the activity of EcoRI endonuclease. To verify further the reliability of this protocol, more control experiments have been performed. First, the active EcoRI was replaced by heat-inactivated EcoRI. It was found from Fig. S1A† that the fluorescence intensity of SGI/H1/H2/ H3 in the presence heat-inactivated EcoRI is almost identical to that of SGI/H1/H2/H3, indicating that the fluorescence amplification is due to the specific cleavage by EcoRI. Then, H*1 hairpin without a specific recognition site of EcoRI endonuclease was chosen as a negative control. It is clear from curve e to f in Fig. S1B† that the fluorescence intensity of SGI was unchanged with the addition of EcoRI. It is obvious that the increase of SGI fluorescence is mainly ascribed to specific cleavage of H1 by EcoRI and triggers a hybridization chain reaction between H2 and H3 to form a long nicked DNA polymer. Finally, the influence of other endonucleases was studied. As shown in Fig. S1C,† there is a slight change in fluorescence intensity when the SGI/H1/H2/H3 mixture is incu-
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bated with BamHI endonuclease, demonstrating that the specific cleavage of H1-triggered HCR led to an increase in fluorescence intensity. Based on the above results, we can draw a conclusion that this label-free signal amplification protocol can be used to study the activity of endonuclease.
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Assay of gel electrophoresis Gel electrophoresis is a powerful tool for the study of biological interactions due to its unique capacity for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments based on their size and charge. First, the specific cleavage of H1 in the presence of EcoRI was verified by gel electrophoresis. It has been found from Fig. 2A that the band of H1 became shallow when H1 was incubated with EcoRI endonuclease. Meanwhile, two new bands appeared, indicating that H1 was specifically cleaved by EcoRI endonucleases and generated two short fragments (lane 2). In contrast, band of H1 remains unchanged when H1 is incubated with heatinactivated EcoRI endonuclease (lane 3). To study further the specificity of enzymatic cleavage, the interactions between
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EcoRI, H2 and H3 were investigated. Both H2 and H3 could not be cleaved by EcoRI endonuclease, illustrating the superior specificity of EcoRI. Therefore, only H1 can be specifically cleaved by EcoRI because its stem contains a recognition site of EcoRI. Then, the specificity of the HCR was studied. As can be seen from Fig. 2B, the band appeared in lane 1, 2, and 3 corresponding to H1, H2 and H3 respectively. The self-hybridization reaction did not occur when H2 was incubated with H3 (lane 4). Meanwhile, HCR did not occur when H1 was incubated with H2 and H3 (lane 5). That is, H1 itself cannot trigger HCR. However, new bands of long DNA fragments were observed when mixture of H1, H2 and H3 was incubated with EcoRI, accompanied by a shallow band of a mixture of H1, H2 and H3 (lane 6). That is, only the specific DNA cleavage by EcoRI endonuclease can open the hairpin conformation of H1 and release DNA initiator to trigger HCR. The specificity of enzymatic cleavage was further confirmed by replacing EcoRI endonuclease with heat-inactivated EcoRI endonuclease. It is obvious from lane 7 in Fig. 2 that HCR was not triggered without active EcoRI endonuclease. The results of gel electrophoresis strongly support the principle of this HCRbased amplification strategy. Identification of double-stranded polymer formation To confirm further the formation of double-stranded polymer in the presence of EcoRI, circular dichroism (CD) measurements were utilized to monitor the conformation changes of the probe under various conditions. It is clear from Fig. S2† that the CD spectra of hairpins shows a positive band at 275 nm and a negative band at 245 nm.20,21 However, we observed an enhancement of the positive peak around 280 nm and the negative peak around 250 nm when mixture of H1, H2 and H3 was incubated with EcoRI, which suggested the formation of a double-stranded polymer. The CD result is in accordance with electrophoresis, demonstrating that DNA cleavage by EcoRI endonuclease successfully triggered HCR, which can be utilized for the highly specific and sensitive study of this biological reaction. Optimization of assay conditions
Fig. 2 (A) Electrophoresis analysis of specific cleavage of H1 by EcoRI endonuclease: (1) H1, (2) H1 + EcoRI, (3) H1 + inactive-EcoRI, (4) H2, (5) H2 + EcoRI, (6) H3, (7) H3 + EcoRI; (B) specificity investigation of HCR by gel electrophoresis: (1)100 nM H1, (2) 500 nM H2, (3) 500 nM H3, (4) 500 nM H2 + 500 nM H3, (5) 100 nM H1 + 500 nM H2 + 500 nM H3, (6) 100 nM H1+ EcoRI + 500 nM H2 + 500 nM H3, (7) 100 nM H1+ inactive 0.2 U μL−1 EcoRI +500 nM H2 + 500 nM H3.
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Many parameters affect the sensitivity, which should be optimized. First, the influence of the concentration of SYBR Green I on the HCR efficiency was investigated. As shown in Fig. S3(A),† the fluorescence efficiency reached its maximum when the concentration of SGI was 30 μM. Thus, 30 μM SGI was chose as signal probe used throughout the experiment. Fig. S3B and C† depicted the effect of H2 and H3 concentrations on the assay performance. The fluorescence intensity of SGI was enhanced greatly with increasing of concentration of H2 and H3 in the range of 0 to 500 nM. However, the background fluorescence also greatly increased when the concentration of H2 and H3 exceeded 500 nM. Both H2 and H3 have a long double-stranded stem which also can bind SGI to induce an increase in background fluorescence. Therefore, the optimum concentration of H2 and H3 was 500 nM. Finally, the
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reaction time of HCR was studied. Fig. S3D† clearly shows that the fluorescence efficiency increased rapidly in the first 4 h and then remained almost constant, indicating that the reaction equilibrium was reached. To obtain the maximal hybridization efficiency, the reaction time is 4 h.
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Assay of endonuclease activity Endonucleases play a vital role in many biological processes such as replication, repair and recombination of nucleic acids.22,23 EcoRI, which belongs to the class II restriction enzymes, has been proven to be a good model system to study the mechanisms of specific protein–nucleic acid interactions.24,25 Therefore, sensitive detection of endonuclease activity is pivotal in the fields of modern molecular biology and medicinal research. Under the optimal conditions, the sensitivity of this assay was evaluated. As shown in Fig. 3A, the fluorescence intensity increased with increasing EcoRI concentration. From the inset of Fig. 3A we discovered that the proposed method presented a good linear response (R = 0.9954) of fluorescence intensity against the logarithm of EcoRI concentration over the range from 0.001 to 0.5 U µL−1. According to the rule of three times of standard deviation, the detection limit of the method is estimated to be 4.1 × 10−4 U µL−1,
Fig. 3 (A) Fluorescence spectra of SGI in the presence of different concentrations of EcoRI. (Inset) Linear correlation of the fluorescence change vs. logarithmic concentrations of EcoRI in the range of 0.001 to 0.5 U μL−1. (B) Fluorescence spectra of SGI in the presence of different concentrations of BamHI. (Inset) Linear correlation of the fluorescence change vs. logarithmic concentrations of BamHI in the range of 0.005 to 1.2 U μL−1. U is the activity unit of the enzyme.
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which is lower than or comparable to the previous reported assay.26–28 Furthermore, compared with previously reported methods, the proposed method is superior in the linear range of detection.24–27 To verify the practicability of this method, the activity of other endonucleases has also been detected by utilizing the proposed method. BamHI is a type II restriction endonuclease, having the capacity for binding at the recognition sequence 5′GGATTC-3′ and specifically cleaving these sequences between G and G on each DNA strand. Here, the activity of BamHI was measured by the proposed method. It is obvious from Fig. 3B that the change in fluorescence intensity can reflect the activity variation of BamHI. Meanwhile, the activity of BamHI also can be evaluated quantitatively in the range of 0.005 to 1.2 U µL−1. Based on the above results, we confirmed that the proposed method is a sensitive and universal tool for analyzing the activity of restriction endonucleases. Specificity of the assay Selectivity is the pivotal matter for biological assays. To identify the reliability of the proposed scheme, the influence of other common proteins has been studied. Six common proteins, including BamHI, lambda exonuclease (λ exo), T4 polynucleotide kinase (T4 PNK), T4 DNA ligase (T4 ligase), immunoglobin G (IgG) and lysozyme (Lys) were chosen as negative controls. First, the activity of EcoRI was confirmed by using inactive EcoRI instead of EcoRI. A slight increase in fluorescence intensity was observed when EcoRI was replaced by inactive EcoRI (Fig. 4A). This is mainly attributed to the fact that inactive EcoRI could not effectively cleave H1 to release HCR initiator. Then, the influence of BamHI endonuclease was studied. Fig. 4A demonstrated that BamHI caused a slight increase in fluorescence intensity. That is, H1 could not be cleaved effectively by BamHI to release HCR initiator. As we know, the recognition site of restriction endonucleases is highly specific. The stem of H1 contains a specific recognition site of EcoRI endonuclease (5′-d (GAATTC)-3′). However, the recognition site of BamHI endonuclease is located at 5′GGATTC-3′. BamHI thus has no effect on the stability of H1. This once again proves that it is a highly specific and reliable method. Finally, the influence of other common proteins were investigated. Fig. 4A sketched the responses of different proteins by comparing the fluorescence enhancement efficiency ([F − F0]/F0) where F0 and F are the fluorescence intensity of H1/H2/H3/SGI in the absence and presence of the proteins, respectively. It clearly illustrated in Fig. 4A that only EcoRI caused a significant increase in fluorescence intensity, whereas the other proteins failed to cause obvious changes in fluorescence intensity. The influence of other common proteins on the HCR has also been studied by gel electrophoresis analysis. As shown in Fig. 4B, HCR is achieved effectively only when H1, H2 and H3 incubated with EcoRI endonuclease. The gel electrophoresis assay is in perfect accordance with the fluorescence analysis. All these results support solidly the conclusion that it is a highly specific and reliable method for studying biological interactions.
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Fig. 4 The influence of common proteins on the specificity. (A) Bar chart of fluorescence enhancement efficiency in the presence of different proteins. The concentrations of EcoRI, inactive EcoRI, BamHI, λ exo, T4 PNK and T4 ligase are 0.2 U μL−1. The concentrations of IgG and Lys are 1 µM. (B) Electrophoresis analysis of specificity.
Evaluation of inhibition of endonuclease activity Molecules which can suppress the activity of endonucleases have great value in biology and clinical research. Therefore, study of the inhibition of the endonuclease activity is of great theoretical and practical importance in screening nuclease inhibitors and drug discovery. According to previous reports, pyrophosphate (PP) and 5-fluorouracil can inhibit the activity of EcoRI endonuclease.27–30 Therefore, the influence of PP and 5-fluorouracil on the EcoRI activity had been investigated. Fig. 5A and B depicted the effect of PP and 5-fluorouracil on the activity of EcoRI endonuclease, indicating that the activity of EcoRI endonuclease reduced as the concentration of PP and 5-fluorouracil increased. That is, PP and 5-fluorouracil are inhibitors of EcoRI endonuclease. PP as an inorganic salt can combine with the enzyme cofactor Mg2+ to generate a new compound. As a consequence, the activity of EcoRI endonuclease is restrained. Some evidence proved that 5-fluorouracil intercalates between AT base pairs slightly more than GC base pairs.29 In addition, this may cause the recognition site of restriction endonuclease to be unrecognizable, thereby protecting DNA from cleavage. To compare further the inhibition efficiency, the IC50 values of the endonuclease inhibitors were
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Fig. 5 Inhibition of EcoRI activity by different inhibitors. (A) Influence of PP on the activity of EcoRI. The concentration of PP is 0, 0.25, 0.5, 1, 2, and 4 mM, respectively. (B) Influence of 5-fluorouracil on the activity of EcoRI. The concentration of 5-fluorouracil is 0, 0.05, 0.1, 0.15, 0.2 and 0.4 mM, respectively. (C) Bar chart of relative activity of EcoRI in the absence and presence of 0.25 mM endonuclease inhibitors. The concentration of EcoRI is 0.2 U μL−1. (D) Electrophoresis analysis of the influence of the different inhibitors.
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measured. The IC50 value is an important indicator to evaluate the inhibition capacity of different inhibitors, which reflect the concentration of inhibitor required to reduce enzyme activity by 50%. According to the data shown in Fig. 5A and B, the IC50 value of PP and 5-fluorouracil was found to be 1.46 mM and 0.1 mM, respectively, which is consistent with the reported literature value determined by other methods.26–28,30 Fig. 5C clearly showed that 5-fluorouracil as an anticancer drug could inhibit more effectively the activity of EcoRI endonuclease than PP. We also used gel electrophoresis to verify the influence of PP and 5-fluorouracil on the EcoRI activity. As shown in Fig. 5D, the HCR efficiency was reduced upon addition of inhibitors (lane 3 and 4). Furthermore, the inhibitor of BamHI endonuclease had also been studied, as shown in Fig. S4.† The results discussed above proved that this label-free method could be explored to screen nuclease inhibitors as well as evaluate endonuclease activity.
Conclusions In summary, an enzyme-free and label-free amplification strategy has been developed to study the activity and inhibition of endonuclease. Compared with previously reported approaches, it offered a more simple and cost-effective amplification strategy without using other enzymes and external labels. Secondly, high specificity is ensured by using a hairpin oligonucleotide as probe to eliminate the influence of non-specific interactions. Furthermore, it offered a sensitive assay for detection of EcoRI activity by introducing HCR amplification. More importantly, the proposed assay is successfully applied in screening of nuclease inhibitors, which holds great promise for the discovery of anticancer drugs. Furthermore, the application for studying the activity and inhibition of BamHI endonuclease demonstrated that it provided a universal platform for studying biological interactions.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (no. 21075079, 21375086), the Fundamental Research Funds for the Central Universities (GK261001097), the Program for Changjiang Scholars and Innovative Research Team in University (IRT 1070) and the program for Innovative Research Team in Shaanxi Province (no. 2014KCT-28).
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Enzyme-free and label-free signal amplification for monitoring endonuclease activity and inhibition via hybridization chain reaction† Jing Zhang, Zhilu Shi and Yan Jin* A label-free and enzyme-free amplification protocol has been proposed for studying endonuclease activity and inhibition on the basis of the enzyme-digested product triggered hybridization chain reaction (HCR). Three hairpin oligonucleotides were designed as probes which could not open or hybridize with each other at room temperature until the initiator DNA was released by specific enzymatic cleavage in the presence of endonuclease to trigger the hybridization chain reaction. SYBR Green I was chosen as a signal probe which intercalated into the grooves of the nicked double DNA polymer, generating a substantially apparent increase in fluorescence intensity. Once the activity of endonuclease is inhibited by enzyme inhibitors, the efficiency of HCR will be greatly decreased. Therefore, screening of endonuclease inhibitors can be achieved effectively as well as the assay of endonuclease activity. Meanwhile, the assay of endonuclease activity and inhibition achieves a better performance as compared to the previous
Received 13th February 2015, Accepted 22nd March 2015
reports. Importantly, it is a more universal method that can be simply used to study activity and inhibition
DOI: 10.1039/c5an00304k
of other endonucleases by changing the specific recognition site. So, the protocol was proved to be a sensitive and cost-effective approach for studying endonuclease activity and inhibition, and as such, it is
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promising for broad potential application in various biological reactions.
Introduction As a promising amplification technique for ultrasensitive bioanalysis, the hybridization chain reaction has aroused significant attention. HCR is based on the chain reaction of recognition and hybridization events between two hairpin DNA probes, which offer an enzyme-free amplification protocol for rapid detection of specific DNA sequences.1–4 HCR events can be triggered only in the presence of the initiator, and each copy of the initiator can trigger a cascade hybridization event.5–9 In addition, the most attractive advantage of HCR is that it allows for selective and specific extension at room temperature without enzyme-aided amplification. Based on the above advantages, numerous studies utilizing the amplification capability of HCR have been developed for detection of DNA, protein, small molecules and metal ions etc.10–16 For example, Zhang’s group combined HCR with enzyme-amplification to develop a fluorescence method for DNA detection by
Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China. E-mail: [email protected]; Fax: (+) 86-29-81530727 † Electronic supplementary information (ESI) available: Additional figures. See DOI: 10.1039/c5an00304k
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using a biotin-labeled hairpin probe and magnetic beads.17 Tang’s group reported an electrochemical immuno-HCR assay for determination of human IgG with DNA-functionalized gold nanoparticles and antibody-immobilized magnetic beads.18 Tan’s group put forward a fluorescence anisotropy strategy for ATP detection by combined target-triggered HCR with biotinmodified probes.19 Tang’s group constructed an electrochemical method for detection of Pb2+ based on the HCR reaction coupling with Pb2+-specific DNAzyme modified magnetic beads.13 As described above, HCR, by its unique assembly approach, has received widespread attention and application. However, to our knowledge, study on the biological reaction by HCR is rare. Herein, to explore further the application of the HCR technique for bioanalysis, a label-free fluorescence method had been developed for sensitive and accurate assay of endonuclease activity and inhibition by using SYBR Green I (SGI) as a signal probe. Protein–DNA interactions play important roles in life processes, including DNA replication, transcription, recombination and repair. The restriction endonuclease is a well-studied class of DNA-binding proteins and has been an important tool in the development of modern molecular biology. Therefore, the specific cleavage of DNA by restriction endonuclease is chosen as a model to explore the feasibility of studying biological reactions by HCR. In this case, three
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hairpin DNAs were designed and named as H1, H2 and H3 respectively. The initiator of HCR and the recognition site of EcoRI endonucleases are, respectively, embedded in the loop and stem part of H1. Thus, initiator strand can be specifically released when H1 was specifically cleaved by EcoRI endonucleases. Then, the initiator strand propagates a chain reaction of alternating H2 and H3 hybridization to form a nicked double-stranded polymer, leading to a significant increase in the fluorescence intensity. Once the activity of the enzyme was reduced by an inhibitor, the HCR efficiency was greatly affected. Therefore, it can be used to screen the endonuclease inhibitor. The assay of activity and inhibition of other endonucleases demonstrated that it offered a simple, rapid and sensitive platform to study biological process by HCR, which is of great theoretical and practical importance in bioanalysis and drug screening.
Experimental Chemicals EcoRI, BamHI endonucleases, T4 polynucleotide kinase (10 U µL−1) and T4 DNA ligase (5 U µL−1) were purchased from Sangon Biotech Co. (Shanghai, China). Lambda exonuclease (5 U µL−1) was purchased from New England Biolabs (NEB, U.K.). Lysozyme, immunoglobin G (IgG) and SYBR Green I (SGI, 19.7 mM) were purchased from Beijing Dingguo Biotechnology Co., Ltd (Beijing, China). DNA oligonucleotides used in this work are listed as follows: H1 (5′-CGCGCTGCGAATTCAGTCTAGGATTCGGCGTGGGTTAAGAATTCGCAGCGCG-3′), H2 (5′CACGCCGAATCCTAGACTCAAAGTAGTCTAGGATTCGGCGTG-3′), H3 (5′-AGTCTAGGATTCGGCGTGGGTTAACACGCCGAATCCTAGACT -3′), H*1 (5′-CGCGCTGCGGATCCAGTCTAGGATTCGGCGTGGGTTAAGGATCCGCAGCGCG-3′). They were synthesized by Shanghai Sangon Biotechnology Co. (Shanghai, China) and were all purified by reverse-phase highperformance liquid chromatography (HPLC). In the hairpin sequences, loops are italicized and sticky ends are underlined. The H1 oligonucleotide stock solutions were prepared with EcoRI buffer (50 mM Tris-HCl solution pH 7.5, 100 mM NaCl, 10 mM MgCl2). H2 and H3 DNA stock solutions were prepared in buffer: 0.4 M NaCl, 50 mM Na2HPO4 ( pH 7.5). Millipore MilliQ (18 MΩ cm) water was used in all experiments.
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Gel electrophoresis H1, H2 and H3 were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h before use. A 12.5% native polyacrylamide gel was prepared by using 1× TBE buffer (100 mM Tris-HCl, 83 mM boric acid, 1 mM EDTA, pH 8.0). The gel was run at 150 V for 40 min with a loading of 5 μL of each sample into the lanes at room temperature, stained with EB for 30 min. The visualization and photography were performed using a Molecular Imager with the Gel Doc system. Circular dichroism spectroscopy The CD spectra of the hairpin and double-stranded polymer were measured by using a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd, England, UK). CD spectra were recorded using a quartz cell of 1 mm optical path length and an instrument scanning speed of 100 nm min−1 with a response time of 2 s at room temperature. CD spectra were obtained by taking the average of three scans made from 210 to 320 nm. All the DNA samples were dissolved in the buffer (0.4 M NaCl, 50 mM Na2HPO4, pH 7.5) and heated to 90 °C for 5 min, gradually cooled to room temperature, and incubated at 4 °C overnight. The background of the buffer was subtracted from the CD data. Assay of EcoRI endonuclease activity and inhibition All the hairpin oligonucleotides were heated to 95 °C for 2 min and then allowed to cool to room temperature for 1 h before use. 100 nM H1 was mixed with different concentrations of EcoRI endonuclease in 25 µL of EcoRI buffer (50 mM Tris-HCl solution 100 mM NaCl, 10 mM MgCl2, pH 7.5) and incubated at 37 °C for 1 h. After that, the above solution was added into 500 nM H2 and 500 nM H3 in 75 µL of reaction buffer (0.4 M NaCl, 50 mM Na2HPO4, pH 7.5). The total volume of the reaction solution was 100 µL. During this process, the hybridization chain reaction was triggered and progressed to form the long nicked DNA polymers. The inhibitor PP and 5-fluorouracil of EcoRI was incubated with 100 nM H1 in Tris-HCl buffer for 15 min at room temperature. Then, EcoRI was added and the resulting solution was incubated at 37 °C for 60 min. The detection procedure was the same as shown in the aforementioned assay of EcoRI endonuclease activity. All fluorescence measurements of the samples were carried out on a fluorometer F7000 (Hitachi, Japan) with excitation at 487 nm and an emission range from 505 to 600 nm, respectively.
Measurements All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Kyoto, Japan). The vertical electrophoresis system was purchased from Bio-Rad Laboratories, Inc. The Molecular Imager system was purchased from Shanghai Peiqing science & Technology. Co., Ltd (Shanghai, China). Circular Dichroism spectra were measured on a Chirascan Circular Dichroism Spectrometer (Applied Photophysics Ltd, England, UK).
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Results and discussion Sensing mechanism In this work, an enzyme-free method has been developed for assay of endonuclease activity and inhibition based on HCR amplification. As shown in Scheme 1, three hairpin probes were designed, including H1, H2 and H3. The stem of H1 contains the specific recognition site of EcoRI endonuclease
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Scheme 1 Schematic illustration of enzyme-free signal amplification for monitoring endonuclease activity and inhibition by the hybridization chain reaction.
(5′-d (GAATTC)-3′) and its loop can hybridize with the fragment I of H2. Both H2 and H3 have two fragments. The fragment I (blue line) at the 5′ end of H2 is complementary to the fragment I′ (blue line) of H3. The fragment II′ (red line) at the 3′ end of H3 can hybridize with the fragment II (red line) of H2. In general, H1, H2 and H3 could not open or hybridize with each other at room temperature in the absence of EcoRI endonucleases. Upon the addition of EcoRI endonucleases, H1 was specifically cleaved at the stem part and generated two fragments. According to the melting temperature (Tm) measurements, the loop fragment of H1 could not form a stable hairpin at room temperature. So, it acts as a single-stranded initiator to trigger the hybridization chain reaction. The initiator strand paired with the sticky end of H2, which undergoes a strand-displacement interaction to open the hairpin structure of H2. Then, the newly exposed sticky end of H2, fragment II, hybridized with the sticky end of H3 (fragment II′) and opened the hairpin of H3 to expose a sticky end (fragment I′) which is identical to the initiator strand in sequence and hybridized with the sticky end of another H2. In this way, each copy of the loop fragment of H1 can give rise to a chain reaction of hybridization events between alternating H2 and H3 hairpins to form a long double-helix. Once the activity of enzyme was restrained by enzyme inhibitors, the HCR efficiency was corresponding relatively reduced due to the low amount of HCR initiator. Therefore, the activity and inhibition of endonuclease can be sensitively monitored by analyzing the change in fluorescence intensity. Fluorescence analysis has been performed to verify the feasibility of the sensing scheme. As shown in Fig. 1A, the fluorescence of free SGI is weak. However, the fluorescence of SGI obviously increased from curve a to e when it was incubated with H2 and H3, because SGI can effectively bind to the double-stranded stem of hairpin probes. In the presence of EcoRI endonuclease, the fluorescence of SGI was enhanced significantly from curve e to f. That is, the specific cleavage of H1 by EcoRI-endonuclease-triggered HCR to form double-helix DNA polymers caused the intercalation of a large quantity of
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Fig. 1 Fluorescence spectra of SGI at different conditions. (A) (a) SGI, (b) H1 + SGI, (c) H1 + EcoRI + SGI, (d) H2 + H3 + SGI, (e) H1 + H2 + H3 + SGI, (f ) H1 + EcoRI + H2 + H3 + SGI. (B) (a) SGI, (b) H1 + SGI, (c) H1 + 5-fluorouracil + EcoRI + SGI, (d) H2 + H3 + SGI, (e) H1 + H2 + H3 + SGI, (f ) H1 + 5-fluorouracil + EcoRI + H2 + H3 + SGI.
SGI into the grooves of dsDNA. So, the EcoRI endonuclease activity can be studied by monitoring the variance of SGI fluorescence intensity. On the contrary, the fluorescence intensity increased slightly from the curve e to f in Fig. 1B when H1 was incubated with the inhibitors. That is, the HCR efficiency is greatly reduced once the activity of the EcoRI endonuclease is restrained in the presence of inhibitor. So, the preliminary results demonstrated that this label-free amplification strategy is feasible for study of the activity of EcoRI endonuclease. To verify further the reliability of this protocol, more control experiments have been performed. First, the active EcoRI was replaced by heat-inactivated EcoRI. It was found from Fig. S1A† that the fluorescence intensity of SGI/H1/H2/ H3 in the presence heat-inactivated EcoRI is almost identical to that of SGI/H1/H2/H3, indicating that the fluorescence amplification is due to the specific cleavage by EcoRI. Then, H*1 hairpin without a specific recognition site of EcoRI endonuclease was chosen as a negative control. It is clear from curve e to f in Fig. S1B† that the fluorescence intensity of SGI was unchanged with the addition of EcoRI. It is obvious that the increase of SGI fluorescence is mainly ascribed to specific cleavage of H1 by EcoRI and triggers a hybridization chain reaction between H2 and H3 to form a long nicked DNA polymer. Finally, the influence of other endonucleases was studied. As shown in Fig. S1C,† there is a slight change in fluorescence intensity when the SGI/H1/H2/H3 mixture is incu-
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bated with BamHI endonuclease, demonstrating that the specific cleavage of H1-triggered HCR led to an increase in fluorescence intensity. Based on the above results, we can draw a conclusion that this label-free signal amplification protocol can be used to study the activity of endonuclease.
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Assay of gel electrophoresis Gel electrophoresis is a powerful tool for the study of biological interactions due to its unique capacity for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments based on their size and charge. First, the specific cleavage of H1 in the presence of EcoRI was verified by gel electrophoresis. It has been found from Fig. 2A that the band of H1 became shallow when H1 was incubated with EcoRI endonuclease. Meanwhile, two new bands appeared, indicating that H1 was specifically cleaved by EcoRI endonucleases and generated two short fragments (lane 2). In contrast, band of H1 remains unchanged when H1 is incubated with heatinactivated EcoRI endonuclease (lane 3). To study further the specificity of enzymatic cleavage, the interactions between
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EcoRI, H2 and H3 were investigated. Both H2 and H3 could not be cleaved by EcoRI endonuclease, illustrating the superior specificity of EcoRI. Therefore, only H1 can be specifically cleaved by EcoRI because its stem contains a recognition site of EcoRI. Then, the specificity of the HCR was studied. As can be seen from Fig. 2B, the band appeared in lane 1, 2, and 3 corresponding to H1, H2 and H3 respectively. The self-hybridization reaction did not occur when H2 was incubated with H3 (lane 4). Meanwhile, HCR did not occur when H1 was incubated with H2 and H3 (lane 5). That is, H1 itself cannot trigger HCR. However, new bands of long DNA fragments were observed when mixture of H1, H2 and H3 was incubated with EcoRI, accompanied by a shallow band of a mixture of H1, H2 and H3 (lane 6). That is, only the specific DNA cleavage by EcoRI endonuclease can open the hairpin conformation of H1 and release DNA initiator to trigger HCR. The specificity of enzymatic cleavage was further confirmed by replacing EcoRI endonuclease with heat-inactivated EcoRI endonuclease. It is obvious from lane 7 in Fig. 2 that HCR was not triggered without active EcoRI endonuclease. The results of gel electrophoresis strongly support the principle of this HCRbased amplification strategy. Identification of double-stranded polymer formation To confirm further the formation of double-stranded polymer in the presence of EcoRI, circular dichroism (CD) measurements were utilized to monitor the conformation changes of the probe under various conditions. It is clear from Fig. S2† that the CD spectra of hairpins shows a positive band at 275 nm and a negative band at 245 nm.20,21 However, we observed an enhancement of the positive peak around 280 nm and the negative peak around 250 nm when mixture of H1, H2 and H3 was incubated with EcoRI, which suggested the formation of a double-stranded polymer. The CD result is in accordance with electrophoresis, demonstrating that DNA cleavage by EcoRI endonuclease successfully triggered HCR, which can be utilized for the highly specific and sensitive study of this biological reaction. Optimization of assay conditions
Fig. 2 (A) Electrophoresis analysis of specific cleavage of H1 by EcoRI endonuclease: (1) H1, (2) H1 + EcoRI, (3) H1 + inactive-EcoRI, (4) H2, (5) H2 + EcoRI, (6) H3, (7) H3 + EcoRI; (B) specificity investigation of HCR by gel electrophoresis: (1)100 nM H1, (2) 500 nM H2, (3) 500 nM H3, (4) 500 nM H2 + 500 nM H3, (5) 100 nM H1 + 500 nM H2 + 500 nM H3, (6) 100 nM H1+ EcoRI + 500 nM H2 + 500 nM H3, (7) 100 nM H1+ inactive 0.2 U μL−1 EcoRI +500 nM H2 + 500 nM H3.
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Many parameters affect the sensitivity, which should be optimized. First, the influence of the concentration of SYBR Green I on the HCR efficiency was investigated. As shown in Fig. S3(A),† the fluorescence efficiency reached its maximum when the concentration of SGI was 30 μM. Thus, 30 μM SGI was chose as signal probe used throughout the experiment. Fig. S3B and C† depicted the effect of H2 and H3 concentrations on the assay performance. The fluorescence intensity of SGI was enhanced greatly with increasing of concentration of H2 and H3 in the range of 0 to 500 nM. However, the background fluorescence also greatly increased when the concentration of H2 and H3 exceeded 500 nM. Both H2 and H3 have a long double-stranded stem which also can bind SGI to induce an increase in background fluorescence. Therefore, the optimum concentration of H2 and H3 was 500 nM. Finally, the
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reaction time of HCR was studied. Fig. S3D† clearly shows that the fluorescence efficiency increased rapidly in the first 4 h and then remained almost constant, indicating that the reaction equilibrium was reached. To obtain the maximal hybridization efficiency, the reaction time is 4 h.
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Assay of endonuclease activity Endonucleases play a vital role in many biological processes such as replication, repair and recombination of nucleic acids.22,23 EcoRI, which belongs to the class II restriction enzymes, has been proven to be a good model system to study the mechanisms of specific protein–nucleic acid interactions.24,25 Therefore, sensitive detection of endonuclease activity is pivotal in the fields of modern molecular biology and medicinal research. Under the optimal conditions, the sensitivity of this assay was evaluated. As shown in Fig. 3A, the fluorescence intensity increased with increasing EcoRI concentration. From the inset of Fig. 3A we discovered that the proposed method presented a good linear response (R = 0.9954) of fluorescence intensity against the logarithm of EcoRI concentration over the range from 0.001 to 0.5 U µL−1. According to the rule of three times of standard deviation, the detection limit of the method is estimated to be 4.1 × 10−4 U µL−1,
Fig. 3 (A) Fluorescence spectra of SGI in the presence of different concentrations of EcoRI. (Inset) Linear correlation of the fluorescence change vs. logarithmic concentrations of EcoRI in the range of 0.001 to 0.5 U μL−1. (B) Fluorescence spectra of SGI in the presence of different concentrations of BamHI. (Inset) Linear correlation of the fluorescence change vs. logarithmic concentrations of BamHI in the range of 0.005 to 1.2 U μL−1. U is the activity unit of the enzyme.
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which is lower than or comparable to the previous reported assay.26–28 Furthermore, compared with previously reported methods, the proposed method is superior in the linear range of detection.24–27 To verify the practicability of this method, the activity of other endonucleases has also been detected by utilizing the proposed method. BamHI is a type II restriction endonuclease, having the capacity for binding at the recognition sequence 5′GGATTC-3′ and specifically cleaving these sequences between G and G on each DNA strand. Here, the activity of BamHI was measured by the proposed method. It is obvious from Fig. 3B that the change in fluorescence intensity can reflect the activity variation of BamHI. Meanwhile, the activity of BamHI also can be evaluated quantitatively in the range of 0.005 to 1.2 U µL−1. Based on the above results, we confirmed that the proposed method is a sensitive and universal tool for analyzing the activity of restriction endonucleases. Specificity of the assay Selectivity is the pivotal matter for biological assays. To identify the reliability of the proposed scheme, the influence of other common proteins has been studied. Six common proteins, including BamHI, lambda exonuclease (λ exo), T4 polynucleotide kinase (T4 PNK), T4 DNA ligase (T4 ligase), immunoglobin G (IgG) and lysozyme (Lys) were chosen as negative controls. First, the activity of EcoRI was confirmed by using inactive EcoRI instead of EcoRI. A slight increase in fluorescence intensity was observed when EcoRI was replaced by inactive EcoRI (Fig. 4A). This is mainly attributed to the fact that inactive EcoRI could not effectively cleave H1 to release HCR initiator. Then, the influence of BamHI endonuclease was studied. Fig. 4A demonstrated that BamHI caused a slight increase in fluorescence intensity. That is, H1 could not be cleaved effectively by BamHI to release HCR initiator. As we know, the recognition site of restriction endonucleases is highly specific. The stem of H1 contains a specific recognition site of EcoRI endonuclease (5′-d (GAATTC)-3′). However, the recognition site of BamHI endonuclease is located at 5′GGATTC-3′. BamHI thus has no effect on the stability of H1. This once again proves that it is a highly specific and reliable method. Finally, the influence of other common proteins were investigated. Fig. 4A sketched the responses of different proteins by comparing the fluorescence enhancement efficiency ([F − F0]/F0) where F0 and F are the fluorescence intensity of H1/H2/H3/SGI in the absence and presence of the proteins, respectively. It clearly illustrated in Fig. 4A that only EcoRI caused a significant increase in fluorescence intensity, whereas the other proteins failed to cause obvious changes in fluorescence intensity. The influence of other common proteins on the HCR has also been studied by gel electrophoresis analysis. As shown in Fig. 4B, HCR is achieved effectively only when H1, H2 and H3 incubated with EcoRI endonuclease. The gel electrophoresis assay is in perfect accordance with the fluorescence analysis. All these results support solidly the conclusion that it is a highly specific and reliable method for studying biological interactions.
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Fig. 4 The influence of common proteins on the specificity. (A) Bar chart of fluorescence enhancement efficiency in the presence of different proteins. The concentrations of EcoRI, inactive EcoRI, BamHI, λ exo, T4 PNK and T4 ligase are 0.2 U μL−1. The concentrations of IgG and Lys are 1 µM. (B) Electrophoresis analysis of specificity.
Evaluation of inhibition of endonuclease activity Molecules which can suppress the activity of endonucleases have great value in biology and clinical research. Therefore, study of the inhibition of the endonuclease activity is of great theoretical and practical importance in screening nuclease inhibitors and drug discovery. According to previous reports, pyrophosphate (PP) and 5-fluorouracil can inhibit the activity of EcoRI endonuclease.27–30 Therefore, the influence of PP and 5-fluorouracil on the EcoRI activity had been investigated. Fig. 5A and B depicted the effect of PP and 5-fluorouracil on the activity of EcoRI endonuclease, indicating that the activity of EcoRI endonuclease reduced as the concentration of PP and 5-fluorouracil increased. That is, PP and 5-fluorouracil are inhibitors of EcoRI endonuclease. PP as an inorganic salt can combine with the enzyme cofactor Mg2+ to generate a new compound. As a consequence, the activity of EcoRI endonuclease is restrained. Some evidence proved that 5-fluorouracil intercalates between AT base pairs slightly more than GC base pairs.29 In addition, this may cause the recognition site of restriction endonuclease to be unrecognizable, thereby protecting DNA from cleavage. To compare further the inhibition efficiency, the IC50 values of the endonuclease inhibitors were
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Fig. 5 Inhibition of EcoRI activity by different inhibitors. (A) Influence of PP on the activity of EcoRI. The concentration of PP is 0, 0.25, 0.5, 1, 2, and 4 mM, respectively. (B) Influence of 5-fluorouracil on the activity of EcoRI. The concentration of 5-fluorouracil is 0, 0.05, 0.1, 0.15, 0.2 and 0.4 mM, respectively. (C) Bar chart of relative activity of EcoRI in the absence and presence of 0.25 mM endonuclease inhibitors. The concentration of EcoRI is 0.2 U μL−1. (D) Electrophoresis analysis of the influence of the different inhibitors.
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measured. The IC50 value is an important indicator to evaluate the inhibition capacity of different inhibitors, which reflect the concentration of inhibitor required to reduce enzyme activity by 50%. According to the data shown in Fig. 5A and B, the IC50 value of PP and 5-fluorouracil was found to be 1.46 mM and 0.1 mM, respectively, which is consistent with the reported literature value determined by other methods.26–28,30 Fig. 5C clearly showed that 5-fluorouracil as an anticancer drug could inhibit more effectively the activity of EcoRI endonuclease than PP. We also used gel electrophoresis to verify the influence of PP and 5-fluorouracil on the EcoRI activity. As shown in Fig. 5D, the HCR efficiency was reduced upon addition of inhibitors (lane 3 and 4). Furthermore, the inhibitor of BamHI endonuclease had also been studied, as shown in Fig. S4.† The results discussed above proved that this label-free method could be explored to screen nuclease inhibitors as well as evaluate endonuclease activity.
Conclusions In summary, an enzyme-free and label-free amplification strategy has been developed to study the activity and inhibition of endonuclease. Compared with previously reported approaches, it offered a more simple and cost-effective amplification strategy without using other enzymes and external labels. Secondly, high specificity is ensured by using a hairpin oligonucleotide as probe to eliminate the influence of non-specific interactions. Furthermore, it offered a sensitive assay for detection of EcoRI activity by introducing HCR amplification. More importantly, the proposed assay is successfully applied in screening of nuclease inhibitors, which holds great promise for the discovery of anticancer drugs. Furthermore, the application for studying the activity and inhibition of BamHI endonuclease demonstrated that it provided a universal platform for studying biological interactions.
Acknowledgements This work was financially supported by the National Natural Science Foundation of China (no. 21075079, 21375086), the Fundamental Research Funds for the Central Universities (GK261001097), the Program for Changjiang Scholars and Innovative Research Team in University (IRT 1070) and the program for Innovative Research Team in Shaanxi Province (no. 2014KCT-28).
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