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Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe Min Xu, Baoxin Li ⇑ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A label-free and sensitive
A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I dye as signal probe.
fluorescence assay for exonuclease activity is developed. Commercially available SYBR Green I (SG) dye is used as signal probe. This method has a linear detection range from 1 to 200 U/mL with a detection limit of 0.7 U/mL.
a r t i c l e
i n f o
Article history: Received 17 January 2015 Received in revised form 8 June 2015 Accepted 17 June 2015 Available online 22 June 2015 Keywords: Exonuclease Fluorescence SYBR Green I Label-free
a b s t r a c t A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I (SG) dye as signal probe. A proof-of-concept of this assay has been demonstrated by using exonuclease III (Exo III) as a model enzyme. In this assay, double-stranded DNA (dsDNA) can bind SG, resulting in a strong fluorescence signal of SG. Upon the addition of Exo III, dsDNA would be digested, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a linear detection range from 1 U/mL to 200 U/mL with a detection limit of 0.7 U/mL. This label-free approach is selective, simple, convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label. The method not only provides a platform for monitoring activity and inhibition of exonuclease but also shows great potential in biological process researches, drug discovery, and clinic diagnostics. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Exonucleases are enzymes that catalyze the hydrolyzing reaction of a polynucleotide chain, and the hydrolysis reaction can break phosphodiester bonds from either the 30 or the 50 end. 30 ? 50 exonucleases are a class of this enzyme family, which ⇑ Corresponding author. E-mail address: [email protected] (B. Li). http://dx.doi.org/10.1016/j.saa.2015.06.052 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
remove nucleases at 30 terminus from DNA. 30 ? 50 exonuclease plays a key role in many important cellular and physiological processes such as DNA proofreading and repair. 30 ? 50 exonuclease activity is involved in many important biological processes, such as DNA proofreading and repairs [1]. Both over-expression and lack of 30 ? 50 exonuclease activity will cause serious diseases and lead to greater susceptibility to cancers and other diseases under stress conditions [2,3]. Therefore, it is important to assay 30 ? 50 exonuclease activity for the diagnosis and therapy of several diseases.
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
Common approach for detection of 30 ? 50 exonuclease activity is gel electrophoresis-based radiographic assays [4]. However, these assays suffer from high labor intensity and inherent safety problems (e.g., health hazard and waste disposal problems). Fluorescence analysis is convenient, high-throughput, cost-effective and sensitive, and has been applied successfully to the determination of DNA and exonucleases [5–9]. In recent years, a lot of effort has been directed toward the development of fluorescence assays for 30 ? 50 exonuclease activity [10–15]. Most of the reported fluorescence methods for exonuclease activity needed fluorescent dyes to label DNA probes. However, design of specific dye-labeled DNA probes is rather difficult and costly. What is more, in spite of the development of these fluorescence methods, further improvement of the analytical performances is still in urgent need. Accordingly, it remains a challenge in developing simple, sensitive and rapid methods for the determination of exonuclease activity. Herein, we report one label-free and sensitive fluorescence assay for exonuclease activity using commercially available SYBR Green I (SG) dye as signal probe. SG has high specificity for double-stranded DNA (dsDNA) and emits bright fluorescence upon intercalation into dsDNA [16–18]. Escherichia coli exonuclease III (Exo III) is the major apurinic/apyrimidinic DNA-repair nuclease [19], and it has a double strand-specific and nonprocessive 30 ? 50 exodeoxyrebonuclease activity [10,20]. We used Exo III as a model enzyme to demonstrate the feasibility of our method. As illustrated in Scheme 1, one dsDNA probe is used as substrate of Exo III and SG. In the absence of Exo III, dsDNA probe can bind SG, and dsDNA/SG exhibits a strong fluorescence at 520 nm. In the presence of Exo III, dsDNA is hydrolyzed into small fragments, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. Compared with the previous reported works, this strategy does not need any complex DNA sequence design or fluorescence dye label. It is convenient but with high analytical performance. The method is not only meaningful for further research on the disease-related
23
biochemical process but also valuable to the molecular-target therapies and the nucleotide exonuclease-target drug discovery. 2. Materials and methods 2.1. Chemicals and materials Exonuclease III (Exo III) was purchased from Takara Biotechnology Co. Ltd. (Dalian, China). All oligonucleotides designed in this study were commercially synthesized by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and purified by high performance liquid chromatography (HPLC). The sequences of oligonucleotides were 50 -TGC CTA CGA GGA ATT CCA TA-30 (ssDNA1) and 50 -TAT GGA ATT CCT CGT AGG CA-30 (ssDNA2). The oligonucleotide stock solutions (20 lM) were prepared in 10 mM phosphate buffered saline (PBS) (5 mM MgCl2, pH 7.4) and diluted to the desired concentration with the same PBS. Before use, the oligonucleotide solution was heated to 90 °C for 5 min and cooled slowly over a 10 min period to room temperature to unwind the single-strand oligonucleotide. SG dye (10,000) was purchased from Sangon Biotech Shanghai Co., Ltd. (China). All other chemicals were of analytical-reagent grade and obtained from standard reagent suppliers. Millipore Milli-Q water (18 MX cm1) was used in all experiments. 2.2. Procedure for Exo III activity detection To prepare duplex DNA, ssDNA1 and ssDNA2 were dissolved in the reaction buffer (10 mM PBS buffer, pH 7.4, 5 mM Mg2+) and diluted with the reaction buffer to the desired concentration. 50 lL ssDNA1 (10 lM) and 50 lL ssDNA1 (10 lM) were mixed and annealed for 10 min at 90 °C and then gradually cooled to room temperature (ca. 20 °C), and was further incubated for
Scheme 1. Schematic representation of label-free fluorescence method for Exo III activity assay.
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
30 min at room temperature to ensure that the stable dsDNA was obtained. Finally, the obtained dsDNA solution was stored at 4 °C for further use. 50 lL dsDNA (0.05 lM) was mixed with various concentrations of 20 lL Exo III, and the whole mixture was incubated at 37 °C for 1 h to complete specific digestion. Finally, 1 lL of SG (100) and reaction buffer were added to give a final volume of 200 lL. After reaction for 10 min at room temperature, the fluorescence spectra of the final solution were measured by using a Hitachi F-4600 Fluorometer (Tokyo, Japan) at the excitation wavelength of 490 nm. The fluorescence emission intensity at 520 nm was recorded. The Exo III activity was quantified by the fluorescence intensity. In the inhibition experiment, to evaluate the effects of inhibitor on Exo III digestion process, different concentrations EDTA were added to 0.05 lM dsDNA solutions and incubated for a certain time. The following procedures were similar as above.
3. Results and discussion 3.1. Strategy for Exo III activity detection The developed strategy for detecting Exo III activity is demonstrated in Scheme 1. One dsDNA, which is obtained by hybridization between ssDNA1 and ssDNA2, acts as the substrate of this enzyme reaction. Exo III catalyzes the removal of mononucleotides from the 30 -terminus of dsDNA [20]. SG is an asymmetrical cyanine dye, and the fluorescence of free SG is very weak. However, the fluorescence of SG can be dramatically enhanced upon binding to dsDNA while no significant fluorescence change can be observed with its binding to ssDNA or mononucleotides [17,18]. This unique characteristic of SG has laid the foundation for the development of Exo III activity detection herein. In the absence of Exo III, SG dye can intercalate into dsDNA and generate strong fluorescence signal. In the presence of Exo III, dsDNA is degraded specifically from the 30 -terminus to from mononucleotides, and SG emits very weak fluorescence. Therefore, quantitative analysis of Exo III activity can be achieved by monitoring the fluorescence intensity change of SG. In order to verify the feasibility of the proposed strategy, an experiment for proof of principle was performed. As shown in Fig. 1, in the absence of Exo III, the fluorescence intensity of the system was very strong (curve a). Once 200 U/mL Exo III is added into the system, the fluorescence intensity significantly decreases (curve b). When ssDNA or no DNA existed in the sensing system, a rather weak fluorescence signal was observed (curve c, d and e). This demonstrated that SG bound strongly to the dsDNA over ssDNA or mononucleotides [18]. The control experiments showed that Exo III itself did not influence the fluorescence intensity of SG (Fig. S1, Supporting Information). In order to further conform that the fluorescence decrease is due to the addition of active Exo III, a heat-inactivated Exo III was used as the second control. The Exo III was heated at 70 °C for 20 min before incubation with dsDNA substrate. The result showed that the fluorescence intensity of dsDNA-SG system in the presence of the heat-inactivated Exo III was almost same as that of dsDNA-SG system itself (Fig. S2). The results strongly indicated that our proposed strategy could be used to detect Exo III activity. On the other hand, the digestion of dsDNA by Exo III could be monitored by Circular Dichroism (CD) spectroscopy. The CD spectrum of the dsDNA before incubation with Exo III displayed a strong negative peak at 245 nm and an intense positive peak at 280 nm. It is well-documented that the peaks at 240 and 280 nm are characteristic of dsDNA [21]. Upon incubation with Exo III, the characteristic peak of duplex DNA is not significant obvious (Fig. S3). This result indicated that the structure of duplex DNA
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Wavelength (nm) Fig. 1. Measurement results of the sensing system under different conditions: (a) dsDNA + SG; (b) dsDNA + Exo III + SG; (c) ssDNA1 + SG; (d) ssDNA2 + SG; (e) SG. Experimental conditions: 0.05 lM dsDNA, 0.05 lM ssDNA1, 0.05 lM ssDNA2, 1 lL SG (100), 200 U/mL Exo III.
was damaged and Exo III efficiently degrades the initial duplex DNA structure. The result further verified that the observed luminescence decrease of the system was attributed to the digestion of dsDNA by Exo III. 3.2. Optimization of experimental conditions In order to maximize the performance of Exo III activity assay, we investigated the effect of various parameters on the fluorescence response of the system to Exo III. We first optimized the dsDNA concentration. As shown in Fig. 2(A), the signal-to-noise ratio was highest when the concentration of dsDNA concentrations was 0.05 lM. So we chose 0.05 lM dsDNA in the further experiment. Furthermore, the incubation time of dsDNA and Exo III is a crucial parameter for Exo III cleavage process. There is no doubt that high concentration of Exo III can digest dsDNA in a short time. In view of the purpose of our assay is detection of Exo III activity, we investigated the process of fluorescence intensity changes over time at a relative low concentration 50 U/mL (Fig. 2(B)). The assay result indicated that the fluorescence intensity of dsDNA-SG system decreased quickly and nearly unchanged after 50 min. In order to obtain a relative good assay result, we chose the incubated time of Exo III was 60 min in the subsequent experiments. 3.3. Performance of the proposed assay for Exo III activity Under the optimized conditions, experiments were carried out by adding Exo III with different concentrations into the system to examine whether the fluorescence change could be used for Exo III activity quantification. Fig. 3 shows that the fluorescence responses for different Exo III concentrations. It is observed that the fluorescence signals gradually decreased as the concentrations of Exo III varied from 1 U/mL to 200 U/mL. There was a good linear relationship in the Exo III concentrations range from 1 U/mL to 200 U/mL (Fig. S4). The limit of detection (LOD) for Exo III was 0.7 U/mL. The detection limit was relative lower comparable to the recently reported G-quadruplex-based label-free fluorescence method [11]. The selectivity of our approach for Exo III activity assay was evaluated by investing the response of the system to other DNA modifying enzymes such as Exonuclease I (Exo I), T4 polynucleotide kinase (T4 PNK) and Bst DNA Polymerase. The results
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
other DNA modifying enzymes, originating from the specific digestion reaction of Exo III on the dsDNA substrate. The cleavage reaction of dsDNA can be prohibited when the Exo III inhibitor is present. Since the cleavage reaction can be followed with this fluorescence method, this fluorescence method can be employed for Exo III inhibitor screening. Ethylenediaminetetraacetic acid (EDTA) is known to be an inhibitor of Exo III [22,23]. In order to demonstrate the potential for screening for inhibitors of nuclease activity, we incubated 25 U/mL Exo III with various concentrations of EDTA. As shown in Fig. S6, increasing the concentration of EDTA resulted in a significant increase in fluorescence intensity, corresponding to a decrease in the percentage cleavage. Exo III activity was completely inhibited at a concentration of 10 mM EDTA. It was clear that our proposed exonuclease activity assay method could be used to screen inhibitors of Exo III.
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4. Conclusions
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In summary, this work demonstrated a label-free fluorescence strategy for the detection of exonuclease activity. The assay relies on a SG fluorescence turn-off mechanism based on digestion of dsDNA by Exo III. Under optimized conditions, this method shows high sensitivity and selectivity for Exo III activity assay. In addition, the method is homogeneous, making it easy to automate by standard robotic manipulation of microwell plates. Given the crucial roles of exonuclease in some biological processes, the method shows great potential in biological process researches, drug discovery, and clinic diagnostics. Acknowledgments
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Wavelength (nm) Fig. 2. (A) Effect of dsDNA concentration on assay sensitivity. (B) Effect of incubation time on the fluorescence intensity. Experimental conditions: 0.05 lM dsDNA, 200 U/mL Exo III, 1 lL SG (100).
This work was supported financially by the National Natural Science Foundation of China (Nos. 21275096, 21475083), Shaanxi Provincial Natural Science Foundation (No. 2013SZS08-Z01), and Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28). Appendix A. Supplementary data
0 U/mL 1 U/mL 10 U/mL 25 U/mL 40 U/mL 50 U/mL 70 U/mL 80 U/mL 100 U/mL 150 U/mL 200 U/mL
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Wavelength (nm) Fig. 3. Fluorescence emission spectra of different concentrations of Exo III. Experimental conditions: 0.05 lM dsDNA, 1 lL SG (100).
(Fig. S5) showed that only Exo III could significantly decrease the luminescence emission of dsDNA-SG system. By comparison, no significant change in emission intensity was observed upon the addition of the other DNA-modifying enzymes. These results indicate that the system displays significant selectivity for Exo III over
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2015.06.052. References [1] I.V. Shevelev, U. Hüebscher, Nat. Rev. Mol. Cell Biol. 3 (2002) 364–376. [2] T.T. Paull, M. Gellert, Mol. Cell 1 (1998) 969–979. [3] D. Kavanagh, D. Spitzer, P.H. Kothari, A. Shaikh, M.K. Liszewski, A. Richards, J.P. Atkinson, Cell Cycle 7 (2008) 1718–1725. [4] M. Brucet, J. Querol-Audí, K. Bertlik, J. Lloberas, I. Fita, A. Celada, Protein Sci. 17 (2008) 2059–2069. [5] K.-H. Leung, H.-Z. He, B. He, H.-J. Zhong, S. Lin, Y.-T. Wang, D.-L. Ma, C.-H. Leung, Chem. Sci. 6 (2015) 2166–2171. [6] L. Lu, D.S.-H. Chan, D.W.J. Kwong, H.-Z. He, C.-H. Leung, D.-L. Ma, Chem. Sci. 5 (2014) 4561–4568. [7] H.-Z. He, K.-H. Leung, W. Wang, D.S.-H. Chan, C.-H. Leung, D.-L. Ma, Chem. Commun. 50 (2014) 5313–5315. [8] D.-L. Ma, L. Lu, S. Lin, B. He, C.-H. Leung, J. Mater. Chem. B 3 (2015) 348–352. [9] C.-H. Leung, H.-J. Zhong, H.-Z. He, L. Lu, D.S.-H. Chan, D.-L. Ma, Chem. Sci. 4 (2013) 3781–3795. [10] X. Su, X.C. Zhu, C. Zhang, X.J. Xiao, M.P. Zhao, Anal. Chem. 84 (2012) 5059– 5065. [11] W. Yang, Y. Ruan, W. Wu, P. Chen, L. Xu, F. Fu, Anal. Bioanal. Chem. 406 (2014) 4535–4540. [12] H.-Z. He, W.-I. Chan, T.-Y. Mak, L.-J. Liu, M. Wang, D.S.-H. Chan, D.-L. Ma, C.-H. Leung, Methods 64 (2013) 218–223. [13] X. Wu, J. Chen, J.X. Zhao, Analyst 139 (2014) 1081–1087. [14] J. Lee, D.H. Min, Analyst 137 (2012) 2024–2026. [15] C.H. Leung, D.S.H. Chan, B.Y.W. Man, C.J. Wang, W. Lam, Y.C. Cheng, W.F. Fong, W.L.W. Hsiao, D.L. Ma, Anal. Chem. 83 (2011) 463–466. [16] L. Kong, J. Xu, Y. Xu, Y. Wang, R. Yuan, Y. Chai, Biosens. Bioelectron. 42 (2013) 193–197.
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[17] C. Lin, Z. Cai, Y. Wang, Z. Zhu, C.J. Yang, X. Chen, Anal. Chem. 86 (2014) 6758– 6762. [18] R. Zhou, C. Xu, J. Dong, G. Wang, Biosens. Bioelectron. 65 (2015) 103–107. [19] M. Gilar, A. Belenky, Y. Budman, D.L. Smisek, A.S. Cohen, J. Chromatogr. B 714 (1998) 13–20. [20] Q. Xu, A. Cao, L.F. Zhang, C.Y. Zhang, Anal. Chem. 84 (2012) 10845–10851.
[21] J. Kypr, I. Kejonvská, D. Rencˇiuk, M. Vorlícˇková, Nucleic Acids Res. 37 (2009) 1713–1725. [22] B. Mouratou, S. Rouyre, S. Pauillac, J.L. Guesdon, Anal. Biochem. 309 (2002) 40– 47. [23] Y. Jiang, J. Tian, S. Chen, Y. Zhao, Y. Wang, S. Zhao, J. Fluorescence 23 (2013) 697–703.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Label-free fluorescence strategy for sensitive detection of exonuclease activity using SYBR Green I as probe Min Xu, Baoxin Li ⇑ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A label-free and sensitive
A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I dye as signal probe.
fluorescence assay for exonuclease activity is developed. Commercially available SYBR Green I (SG) dye is used as signal probe. This method has a linear detection range from 1 to 200 U/mL with a detection limit of 0.7 U/mL.
a r t i c l e
i n f o
Article history: Received 17 January 2015 Received in revised form 8 June 2015 Accepted 17 June 2015 Available online 22 June 2015 Keywords: Exonuclease Fluorescence SYBR Green I Label-free
a b s t r a c t A label-free and sensitive fluorescence assay for exonuclease activity is developed using commercially available SYBR Green I (SG) dye as signal probe. A proof-of-concept of this assay has been demonstrated by using exonuclease III (Exo III) as a model enzyme. In this assay, double-stranded DNA (dsDNA) can bind SG, resulting in a strong fluorescence signal of SG. Upon the addition of Exo III, dsDNA would be digested, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. This method has a linear detection range from 1 U/mL to 200 U/mL with a detection limit of 0.7 U/mL. This label-free approach is selective, simple, convenient and cost-efficient without any complex DNA sequence design or fluorescence dye label. The method not only provides a platform for monitoring activity and inhibition of exonuclease but also shows great potential in biological process researches, drug discovery, and clinic diagnostics. Ó 2015 Elsevier B.V. All rights reserved.
1. Introduction Exonucleases are enzymes that catalyze the hydrolyzing reaction of a polynucleotide chain, and the hydrolysis reaction can break phosphodiester bonds from either the 30 or the 50 end. 30 ? 50 exonucleases are a class of this enzyme family, which ⇑ Corresponding author. E-mail address: [email protected] (B. Li). http://dx.doi.org/10.1016/j.saa.2015.06.052 1386-1425/Ó 2015 Elsevier B.V. All rights reserved.
remove nucleases at 30 terminus from DNA. 30 ? 50 exonuclease plays a key role in many important cellular and physiological processes such as DNA proofreading and repair. 30 ? 50 exonuclease activity is involved in many important biological processes, such as DNA proofreading and repairs [1]. Both over-expression and lack of 30 ? 50 exonuclease activity will cause serious diseases and lead to greater susceptibility to cancers and other diseases under stress conditions [2,3]. Therefore, it is important to assay 30 ? 50 exonuclease activity for the diagnosis and therapy of several diseases.
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
Common approach for detection of 30 ? 50 exonuclease activity is gel electrophoresis-based radiographic assays [4]. However, these assays suffer from high labor intensity and inherent safety problems (e.g., health hazard and waste disposal problems). Fluorescence analysis is convenient, high-throughput, cost-effective and sensitive, and has been applied successfully to the determination of DNA and exonucleases [5–9]. In recent years, a lot of effort has been directed toward the development of fluorescence assays for 30 ? 50 exonuclease activity [10–15]. Most of the reported fluorescence methods for exonuclease activity needed fluorescent dyes to label DNA probes. However, design of specific dye-labeled DNA probes is rather difficult and costly. What is more, in spite of the development of these fluorescence methods, further improvement of the analytical performances is still in urgent need. Accordingly, it remains a challenge in developing simple, sensitive and rapid methods for the determination of exonuclease activity. Herein, we report one label-free and sensitive fluorescence assay for exonuclease activity using commercially available SYBR Green I (SG) dye as signal probe. SG has high specificity for double-stranded DNA (dsDNA) and emits bright fluorescence upon intercalation into dsDNA [16–18]. Escherichia coli exonuclease III (Exo III) is the major apurinic/apyrimidinic DNA-repair nuclease [19], and it has a double strand-specific and nonprocessive 30 ? 50 exodeoxyrebonuclease activity [10,20]. We used Exo III as a model enzyme to demonstrate the feasibility of our method. As illustrated in Scheme 1, one dsDNA probe is used as substrate of Exo III and SG. In the absence of Exo III, dsDNA probe can bind SG, and dsDNA/SG exhibits a strong fluorescence at 520 nm. In the presence of Exo III, dsDNA is hydrolyzed into small fragments, and SG emits very weak fluorescence. Thus, Exo III activity can be facilely measured with a simple fluorescence reader. Compared with the previous reported works, this strategy does not need any complex DNA sequence design or fluorescence dye label. It is convenient but with high analytical performance. The method is not only meaningful for further research on the disease-related
23
biochemical process but also valuable to the molecular-target therapies and the nucleotide exonuclease-target drug discovery. 2. Materials and methods 2.1. Chemicals and materials Exonuclease III (Exo III) was purchased from Takara Biotechnology Co. Ltd. (Dalian, China). All oligonucleotides designed in this study were commercially synthesized by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and purified by high performance liquid chromatography (HPLC). The sequences of oligonucleotides were 50 -TGC CTA CGA GGA ATT CCA TA-30 (ssDNA1) and 50 -TAT GGA ATT CCT CGT AGG CA-30 (ssDNA2). The oligonucleotide stock solutions (20 lM) were prepared in 10 mM phosphate buffered saline (PBS) (5 mM MgCl2, pH 7.4) and diluted to the desired concentration with the same PBS. Before use, the oligonucleotide solution was heated to 90 °C for 5 min and cooled slowly over a 10 min period to room temperature to unwind the single-strand oligonucleotide. SG dye (10,000) was purchased from Sangon Biotech Shanghai Co., Ltd. (China). All other chemicals were of analytical-reagent grade and obtained from standard reagent suppliers. Millipore Milli-Q water (18 MX cm1) was used in all experiments. 2.2. Procedure for Exo III activity detection To prepare duplex DNA, ssDNA1 and ssDNA2 were dissolved in the reaction buffer (10 mM PBS buffer, pH 7.4, 5 mM Mg2+) and diluted with the reaction buffer to the desired concentration. 50 lL ssDNA1 (10 lM) and 50 lL ssDNA1 (10 lM) were mixed and annealed for 10 min at 90 °C and then gradually cooled to room temperature (ca. 20 °C), and was further incubated for
Scheme 1. Schematic representation of label-free fluorescence method for Exo III activity assay.
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
30 min at room temperature to ensure that the stable dsDNA was obtained. Finally, the obtained dsDNA solution was stored at 4 °C for further use. 50 lL dsDNA (0.05 lM) was mixed with various concentrations of 20 lL Exo III, and the whole mixture was incubated at 37 °C for 1 h to complete specific digestion. Finally, 1 lL of SG (100) and reaction buffer were added to give a final volume of 200 lL. After reaction for 10 min at room temperature, the fluorescence spectra of the final solution were measured by using a Hitachi F-4600 Fluorometer (Tokyo, Japan) at the excitation wavelength of 490 nm. The fluorescence emission intensity at 520 nm was recorded. The Exo III activity was quantified by the fluorescence intensity. In the inhibition experiment, to evaluate the effects of inhibitor on Exo III digestion process, different concentrations EDTA were added to 0.05 lM dsDNA solutions and incubated for a certain time. The following procedures were similar as above.
3. Results and discussion 3.1. Strategy for Exo III activity detection The developed strategy for detecting Exo III activity is demonstrated in Scheme 1. One dsDNA, which is obtained by hybridization between ssDNA1 and ssDNA2, acts as the substrate of this enzyme reaction. Exo III catalyzes the removal of mononucleotides from the 30 -terminus of dsDNA [20]. SG is an asymmetrical cyanine dye, and the fluorescence of free SG is very weak. However, the fluorescence of SG can be dramatically enhanced upon binding to dsDNA while no significant fluorescence change can be observed with its binding to ssDNA or mononucleotides [17,18]. This unique characteristic of SG has laid the foundation for the development of Exo III activity detection herein. In the absence of Exo III, SG dye can intercalate into dsDNA and generate strong fluorescence signal. In the presence of Exo III, dsDNA is degraded specifically from the 30 -terminus to from mononucleotides, and SG emits very weak fluorescence. Therefore, quantitative analysis of Exo III activity can be achieved by monitoring the fluorescence intensity change of SG. In order to verify the feasibility of the proposed strategy, an experiment for proof of principle was performed. As shown in Fig. 1, in the absence of Exo III, the fluorescence intensity of the system was very strong (curve a). Once 200 U/mL Exo III is added into the system, the fluorescence intensity significantly decreases (curve b). When ssDNA or no DNA existed in the sensing system, a rather weak fluorescence signal was observed (curve c, d and e). This demonstrated that SG bound strongly to the dsDNA over ssDNA or mononucleotides [18]. The control experiments showed that Exo III itself did not influence the fluorescence intensity of SG (Fig. S1, Supporting Information). In order to further conform that the fluorescence decrease is due to the addition of active Exo III, a heat-inactivated Exo III was used as the second control. The Exo III was heated at 70 °C for 20 min before incubation with dsDNA substrate. The result showed that the fluorescence intensity of dsDNA-SG system in the presence of the heat-inactivated Exo III was almost same as that of dsDNA-SG system itself (Fig. S2). The results strongly indicated that our proposed strategy could be used to detect Exo III activity. On the other hand, the digestion of dsDNA by Exo III could be monitored by Circular Dichroism (CD) spectroscopy. The CD spectrum of the dsDNA before incubation with Exo III displayed a strong negative peak at 245 nm and an intense positive peak at 280 nm. It is well-documented that the peaks at 240 and 280 nm are characteristic of dsDNA [21]. Upon incubation with Exo III, the characteristic peak of duplex DNA is not significant obvious (Fig. S3). This result indicated that the structure of duplex DNA
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Wavelength (nm) Fig. 1. Measurement results of the sensing system under different conditions: (a) dsDNA + SG; (b) dsDNA + Exo III + SG; (c) ssDNA1 + SG; (d) ssDNA2 + SG; (e) SG. Experimental conditions: 0.05 lM dsDNA, 0.05 lM ssDNA1, 0.05 lM ssDNA2, 1 lL SG (100), 200 U/mL Exo III.
was damaged and Exo III efficiently degrades the initial duplex DNA structure. The result further verified that the observed luminescence decrease of the system was attributed to the digestion of dsDNA by Exo III. 3.2. Optimization of experimental conditions In order to maximize the performance of Exo III activity assay, we investigated the effect of various parameters on the fluorescence response of the system to Exo III. We first optimized the dsDNA concentration. As shown in Fig. 2(A), the signal-to-noise ratio was highest when the concentration of dsDNA concentrations was 0.05 lM. So we chose 0.05 lM dsDNA in the further experiment. Furthermore, the incubation time of dsDNA and Exo III is a crucial parameter for Exo III cleavage process. There is no doubt that high concentration of Exo III can digest dsDNA in a short time. In view of the purpose of our assay is detection of Exo III activity, we investigated the process of fluorescence intensity changes over time at a relative low concentration 50 U/mL (Fig. 2(B)). The assay result indicated that the fluorescence intensity of dsDNA-SG system decreased quickly and nearly unchanged after 50 min. In order to obtain a relative good assay result, we chose the incubated time of Exo III was 60 min in the subsequent experiments. 3.3. Performance of the proposed assay for Exo III activity Under the optimized conditions, experiments were carried out by adding Exo III with different concentrations into the system to examine whether the fluorescence change could be used for Exo III activity quantification. Fig. 3 shows that the fluorescence responses for different Exo III concentrations. It is observed that the fluorescence signals gradually decreased as the concentrations of Exo III varied from 1 U/mL to 200 U/mL. There was a good linear relationship in the Exo III concentrations range from 1 U/mL to 200 U/mL (Fig. S4). The limit of detection (LOD) for Exo III was 0.7 U/mL. The detection limit was relative lower comparable to the recently reported G-quadruplex-based label-free fluorescence method [11]. The selectivity of our approach for Exo III activity assay was evaluated by investing the response of the system to other DNA modifying enzymes such as Exonuclease I (Exo I), T4 polynucleotide kinase (T4 PNK) and Bst DNA Polymerase. The results
M. Xu, B. Li / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 151 (2015) 22–26
other DNA modifying enzymes, originating from the specific digestion reaction of Exo III on the dsDNA substrate. The cleavage reaction of dsDNA can be prohibited when the Exo III inhibitor is present. Since the cleavage reaction can be followed with this fluorescence method, this fluorescence method can be employed for Exo III inhibitor screening. Ethylenediaminetetraacetic acid (EDTA) is known to be an inhibitor of Exo III [22,23]. In order to demonstrate the potential for screening for inhibitors of nuclease activity, we incubated 25 U/mL Exo III with various concentrations of EDTA. As shown in Fig. S6, increasing the concentration of EDTA resulted in a significant increase in fluorescence intensity, corresponding to a decrease in the percentage cleavage. Exo III activity was completely inhibited at a concentration of 10 mM EDTA. It was clear that our proposed exonuclease activity assay method could be used to screen inhibitors of Exo III.
4.5 4.0
A
3.5 3.0
F0/F
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0.025
0.01
0.05
0.50
0.075
25
Concentration of dsDNA (μM)
4. Conclusions
1600
0 min 2 min 4 min 6 min 8 min 10 min 20 min 30 min 40 min 50 min 60 min 70 min
B Fluorescence intensity (a.u.)
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In summary, this work demonstrated a label-free fluorescence strategy for the detection of exonuclease activity. The assay relies on a SG fluorescence turn-off mechanism based on digestion of dsDNA by Exo III. Under optimized conditions, this method shows high sensitivity and selectivity for Exo III activity assay. In addition, the method is homogeneous, making it easy to automate by standard robotic manipulation of microwell plates. Given the crucial roles of exonuclease in some biological processes, the method shows great potential in biological process researches, drug discovery, and clinic diagnostics. Acknowledgments
200 0 520
540
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Wavelength (nm) Fig. 2. (A) Effect of dsDNA concentration on assay sensitivity. (B) Effect of incubation time on the fluorescence intensity. Experimental conditions: 0.05 lM dsDNA, 200 U/mL Exo III, 1 lL SG (100).
This work was supported financially by the National Natural Science Foundation of China (Nos. 21275096, 21475083), Shaanxi Provincial Natural Science Foundation (No. 2013SZS08-Z01), and Program for Innovative Research Team in Shaanxi Province (No. 2014KCT-28). Appendix A. Supplementary data
0 U/mL 1 U/mL 10 U/mL 25 U/mL 40 U/mL 50 U/mL 70 U/mL 80 U/mL 100 U/mL 150 U/mL 200 U/mL
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Fluorescence intensity (a.u.)
1400 1200 1000 800 600 400 510
520
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Wavelength (nm) Fig. 3. Fluorescence emission spectra of different concentrations of Exo III. Experimental conditions: 0.05 lM dsDNA, 1 lL SG (100).
(Fig. S5) showed that only Exo III could significantly decrease the luminescence emission of dsDNA-SG system. By comparison, no significant change in emission intensity was observed upon the addition of the other DNA-modifying enzymes. These results indicate that the system displays significant selectivity for Exo III over
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