Analytical Biochemistry 468 (2015) 34–38
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters Lihui Wang, Keke Ma, Yaodong Zhang ⇑ Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 9 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: S1 nuclease Silver nanoclusters Fluorometry Enzyme activity Enzyme inhibitor
a b s t r a c t S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12–Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5 108 U ll1 (signal/noise = 3) can be determined with a linear range of 5 107 to 1 103 U ll1. The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis. Ó 2014 Elsevier Inc. All rights reserved.
S1 nuclease hydrolyzes single-stranded DNA (ssDNA)1 or RNA into 50 -phosphomononucleotide and 50 -phosphooligonucleotide. This enzyme has been widely used in various applications such as in the determination of nucleic acid structure, the quantitation of nucleic acid hybridization, mapping mutations, detection of gaps in duplex DNA, and studying the interactions of DNA with various intercalating agents [1–3]. S1 nuclease has been particularly used to probe the disruption of the DNA structure by numerous carcinogens and antimitotic drugs [4]. Therefore, detecting S1 nuclease activity and screening for its potential inhibitors is substantially important. Numerous traditional methods have been used to measure the nuclease activity [5]. Although these methods are generally accurate, their routine use in laboratory is restricted because of their laborious and complicated conjugated chemistries. Recently, a number of colorimetric [6], electrochemical [7,8], and fluorescent [3,9,10] assays for S1 nuclease detection have been developed. In ⇑ Corresponding author at: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China. Fax: +86 29 81530727. E-mail address: [email protected] (Y. Zhang). 1 Abbreviations used: ssDNA, single-stranded DNA; NC, nanocluster; Cu, copper; NP, nanoparticle; dC12, polycytosine oligonucleotide; Ag, silver; UV–Vis, ultraviolet– visible; PB, phosphate buffer. http://dx.doi.org/10.1016/j.ab.2014.09.011 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
addition, advanced materials, such as gold nanoparticles [6], carbon nanotubes [11], perylene derivatives [3], distyrylanthracene [10], and cationic polythiophene derivative [12], have been used for the construction of novel assay strategies for S1 nuclease. However, these strategies have specific drawbacks such as low detection sensitivity, expensive materials, and time-consuming assay procedures. Therefore, developing a highly sensitive and facile method for detecting S1 nuclease activity and screening for its potential inhibitors is substantially important. Noble metal nanoclusters (NCs) have been used in various applications, such as bioimaging, biosensing, and specific protein detection, because of their ultrafine size, nontoxicity, and good biocompatibility [13–21]. NCs can be routinely prepared in aqueous solutions with a number of thiolated or polymeric templates, including thiolated compounds [22], DNAs [23,24], proteins [25], and dendrimers [26,27]. DNA is a particularly interesting ligand for preparing fluorescent metal NCs and developing biosensors [28]. For the detection of S1 nuclease activity, two interesting DNA-templated NC methods, namely the fluorometric assay based on double-strand [29] or single-strand [30] DNA-templated copper (Cu) NCs/NPs (nanoparticles), have been recently developed. However, the relatively low quantum yield of Cu NCs/NPs (e.g., a value of 0.068 for T30-templated Cu NCs/NPs) [30] indicates that the detection sensitivity of these methods could be further improved.
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
In this study, polycytosine oligonucleotide (dC12) was used as both the S1 nuclease substrate and template for the formation of highly fluorescent dC12–Ag (silver) NCs. S1 nuclease degrades dC12 to nucleotide fragments, thereby resulting in a reduction in fluorescence. Thus, a label-free and highly sensitive method for S1 nuclease activity assay was developed. Materials and methods Materials and apparatus The S1 nuclease was provided by TaKaRa Biotechnology (Dalian, China). Oligonucleotides with dC12 were synthesized and purified by Sangon Biotechnology (Shanghai, China). The oligonucleotide stock solutions (200 lM) were prepared with deionized water and kept frozen at 20 °C. The RPMI 1640 cell medium was also purchased from Sangon Biotechnology. All other chemicals were of analytical grade and used without further treatment. Deionized water was prepared using a Millipore Milli-Q water purification system (18.2 MO). The ultraviolet–visible (UV–Vis) and fluorescence spectra of DNA–Ag NCs were obtained using a PerkinElmer Lambda 35 spectrometer and a PerkinElmer LS 55 spectrometer, respectively. Mass spectra were acquired in negative ion mode with 2.5-kV needle and 40-V cone voltages. Determination of S1 nuclease and inhibitory detection In total, 100 ll of the mixtures containing 20 lM dC12 and various concentrations of S1 nuclease ranging from 0.0 U ll1 to 5 102 U ll1 was incubated in an enzyme reaction buffer (30 mM CH3COONa, 100 mM NaNO3, and 1 mM Zn(NO3)2, pH 4.6) at 37 °C for 0.5 h in the PCR (polymerase chain reaction) tube. After enzyme digestion, the mixture was heated at 95 °C for 10 min to terminate the cleavage reaction. The solutions of sodium phosphate buffer (PB; 5 mM, pH 7.0, 92 ll) and AgNO3 (2 mM, 6 ll) were then separately introduced and further incubated in an ice bath for 15 min. Lastly, freshly prepared NaBH4 (5 mM, 2.5 ll) by dissolving NaBH4 powder in deionized water was added to induce fluorescent DNA–Ag NC formation. After being kept in the dark for 2 h at room temperature, the mixtures were diluted with PB for subsequent fluorescence measurements. The 10-fold amount (1 102 U/ll) of EcoRI, acid and alkaline phosphatase, ExoIII, and Escherichia coli ligase in the mixture was used to evaluate the specificity of the fluorometric assay to the S1 nuclease. The inhibition experiments were the same as for the above procedure except for the 15-min preferential incubation of the inhibitor with the S1 nuclease before the addition of dC12.
35
Results and discussion Detection mechanism of the method Scheme 1 illustrates the principle of the Ag NC-based fluorescent sensing system for S1 nuclease. The assay mechanism was based on function of dC12 as both the S1 nuclease substrate and template for the stabilization of Ag NCs. The oligonucleotide dC12 was selected as substrate because the dC12-templated silver clusters exhibit excellent fluorescence properties and have been well characterized by Ritchie and coworkers [31] and successfully used in bioanalysis [32]. In the absence of S1 nuclease, the strong fluorescent dC12–Ag NCs could be formed through the reduction of Ag+ by NaBH4. However, dC12 could be digested into mono- and/ or small fragments by the S1 nuclease, which results in the failed formation of Ag NCs because of the lack of a suitable template. Thus, S1 nuclease activity can be successfully determined through the change in the fluorescence of dC12–Ag NCs. Characteristics of Ag NCs and feasibility of the method The dC12–Ag NCs were synthesized according to the procedures in the literature [31,32]. To confirm the formation of the fluorescent dC12–Ag NCs, both UV–Vis and fluorescence spectra were analyzed. The absorbance spectra (Fig. 1, curve a) show two dominant peaks at maximum absorbance wavelengths of 443
Scheme 1. Schematic diagram of S1 nuclease activity assay based on dC12–Ag NCs.
Dynamic detection of S1 nuclease A series of 100-ll reaction mixtures containing a fixed concentration of 20 lM dC12 and 5 103 U ll1 S1 nuclease were incubated at 37 °C for 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, and 30 min. After incubation for a predetermined period, the following experiments were performed according to the aforementioned procedure. Detection of S1 nuclease in complex samples To test the practicality of the proposed strategy, the target from the complex samples was analyzed according to the methods presented in the literature [30]. Different amounts of S1 nuclease were spiked into RPMI 1640 cell medium, in which 1 mM zinc nitrate was added to protect the enzyme activity. The S1 nuclease was subsequently detected via the proposed method, and the recovery value was determined.
Fig.1. UV–Vis (a) and fluorescence spectra of dC12–Ag NC probe: excitation curve (b), emission curve (c), incubation with inactivated S1 nuclease (d), and incubation with activated S1 nuclease (e). Maximum wavelengths of excitation and emission are 555 and 611 nm, respectively.
36
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
and 550 nm. The strongest fluorescence emission peak at 611 nm (curve c) was obtained on excitation at 555 nm (curve b). The oligonucleotide dC12 is bound with several silver atoms, as demonstrated in electrospray ionization mass spectra (see Fig. S1 in the online supplementary material). These results are similar to those reported in the literature [31] and indicate the formation of dC12–Ag NCs. To verify the feasibility of the designed assay strategy, the enzymatic cleavage reaction of S1 nuclease was investigated. dC12 with a concentration of 20 lM was incubated with 5 102 U ll1 S1 nuclease and with the same amount of inactivated S1 nuclease. After cleavage reaction at 37 °C for 30 min and subsequent heating at 95 °C for 10 min to terminate the reaction, the PB, AgNO3, and NaBH4 were added to induce fluorescent Ag NC formation. The resulting reaction solutions were then diluted and analyzed via fluorescence measurements. Fig. 1 shows that when dC12 was incubated with inactivated S1 nuclease and heated at 95 °C for 10 min to be denatured, the fluorescence intensity of dC12–Ag NCs (curve d) was almost the same as that without S1 nuclease (curve c). By contrast, the fluorescence intensity of the prepared dC12–Ag NCs significantly decreased (curve e) in the presence of active S1 nuclease. Therefore, the fluorescence decreased because of dC12 digestion by the S1 nuclease. The dynamic cleavage reaction of dC12 by the S1 nuclease was subsequently investigated. At cleavage times between 0 and 30 min, the fluorescence intensity at 611 nm of the obtained dC12–Ag NCs gradually decreased as a function of time (Fig. 2). The fluorescence rapidly decreased during the first 5 min and remained almost constant after 20 min, thereby demonstrating that the detection system can quickly respond to the S1 nuclease. Assay of nuclease activity To evaluate the assay performance, various amounts of S1 nuclease were added into the dC12 solutions and incubated for 0.5 h. The mixtures were then heated to 95 °C for 10 min to terminate the cleavage reaction and were used to prepare dC12–Ag NCs. Fig. 3 shows the obtained fluorescence spectra of the prepared dC12–Ag NCs. The fluorescence intensity at 611 nm decreased as a function of S1 nuclease concentration. The inset in Fig. 3 illustrates the linear relationship (R2 = 0.9966) between the fluorescence intensity ratio (F0/F) of DNA–Ag NCs at 611 nm and the S1 nuclease concentration ranging from 5 107 to 1 103
Fig.3. Fluorescence spectra of detection system with different S1 nuclease concentrations. Curves from a to k refer to 0.0, 5.0 108, 5.0 107, 5.0 106, 5.0 105, 2.5 104, 5.0 104, 1.0 103, 2.5 103, 5.0 103, and 5.0 102 U ll1, respectively. The inset shows the linear relationship of fluorescence intensity ratios (F0/F) at 611 nm versus S1 nuclease concentrations ranging from 5.0 107 to 1.0 103 U ll1.
U ll1, where F0 and F are the fluorescence intensities of the sensor solution in the absence and presence of the S1 nuclease, respectively. The detection limit was calculated to be 5 108 U ll1 according to the rule of 3 times the standard deviation over the blank response. The assay specificity was also tested by examining the interference of other common proteins or enzymes on the assay for S1 nuclease. As described in Materials and Methods, EcoRI, acid and alkaline phosphatase, ExoIII, and E. coli ligase were studied. Only S1 nuclease remarkably reduced the fluorescence intensities of the proteins or enzymes at the same conditions, and the values are almost equal to the maximum fluorescence emission intensity even though their concentrations were 10 times higher than that of the S1 nuclease (see Fig. S2 in supplementary material). These results indicate that the proposed method has a high selectivity toward nuclease. Inhibition assay of S1 nuclease Several studies have proven that the disruption of the S1 nuclease function is essential for many biological processes. Thus, the pharmacological inhibition of this enzyme provides a broad spectrum of therapeutic applications. In this study, we demonstrated the capacity of the developed method for detecting the inhibitors of the S1 nuclease by using pyrophosphate as the model inhibitor [11]. Fig. 4 shows that the relative activity of the S1 nuclease decreased with increasing pyrophosphate concentrations, and the concentration that caused a 50% fluorescence recovery is approximately 0.21 mM. The IC50 value was different from those reported before [1,11]. However, this is understandable given that the IC50 values usually depend on the measurement conditions such as method for determination, enzyme concentration, and pH [33]. These results demonstrate that the developed method has potential applications in screening for S1 nuclease-targeted drugs, which is important for antibiotics and anticancer therapeutics. Detection of S1 nuclease activity in complex samples
Fig.2. Fluorescence spectra of dC12–Ag NCs in the presence of S1 nuclease incubated for different periods. Inset: Time-dependent fluorescent changes at 611 nm. The S1 nuclease concentration is 1 103 U ll1.
To test the practicality of the proposed strategy, we chose the RPMI 1640 cell medium, into which five different S1 nuclease
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
37
References
Fig.4. Inhibitory effects of pyrophosphate on S1 nuclease.
Table 1 Measurement of S1 nuclease activity in RPMI 1640 cell medium. Added (103 U ll1)
Detected (103 U ll1)
Recovery (%)
RSD (%, n = 3)
0.050 0.10 0.50 5.0 10.0
0.047 0.097 0.49 4.8 9.5
94 97 98 96 95
6.7 5.4 7.9 5.2 8.1
Note. RSD, relative standard deviation.
concentrations (Table1) were spiked as the complex fluids. Table 1 shows that the obtained recoveries and reproducibility based on our strategy were desirable. These results indicate that the proposed method has considerable potential in practical applications for S1 nuclease detection with excellent accuracy and reliability. Conclusions We have provided a novel ultrasensitive and label-free fluorometric method for monitoring the cleavage of ssDNA by S1 nuclease and screening of enzyme inhibitors. The method depends on the function of dC12 as both the S1 nuclease substrate and Ag NC template. The significant decrease in the DNA–Ag NC emission could be used to monitor the DNA cleavage by the S1 nuclease. The detection limit is 5 108 U ll1, which is better than those obtained from previously reported methods [30]. The developed method not only is facile because its operation does not require any complex labeling of DNA or sophisticated experimental techniques but also offers a convenient strategy for a homogeneous and rapid detection with high sensitivity. Acknowledgments This work was supported by the National Natural Science Foundation of China (21275097) and the Fundamental Research Fund for the Central Universities (GK201303001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.09.011.
[1] R.C. Wiegand, G.N. Godson, C.M. Radding, Specificity of the S1 nuclease from Aspergillus oryzae, J. Biol. Chem. 250 (1975) 8848–8855. [2] N.A. Desai, V. Shankar, Single-strand-specific nucleases, FEMS Microbiol. Rev. 26 (2003) 457–491. [3] X. Yang, F. Pu, J. Ren, X. Qu, DNA-templated ensemble for label-free and realtime fluorescence turn-on detection of enzymatic/oxidative cleavage of singlestranded DNA, Chem. Commun. 47 (2011) 8133–8135 (Camb.). [4] J.L. Butour, A.M. Mazard, C. Vieussens, N.P. Johnson, Kinetic studies of the hydrolysis of platinum–DNA complexes by nuclease S1, Chem. Biol. Interact. 73 (1990) 195–205. [5] B. Mouratou, S. Rouyre, S. Pauillac, J.L. Guesdon, Development of nonradioactive microtiter plate assays for nuclease activity, Anal. Biochem. 309 (2002) 40–47. [6] R. Cao, B. Li, Y. Zhang, Z. Zhang, Naked-eye sensitive detection of nuclease activity using positively-charged gold nanoparticles as colorimetric probes, Chem. Commun. 47 (2011) 12301–12303 (Camb.). [7] J. Ding, W. Qin, Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode, Biosens. Bioelectron. 47 (2013) 559–565. [8] S.C. Hillier, C.G. Frost, A.T. Jenkins, H.T. Braven, R.W. Keay, S.E. Flower, J.M. Clarkson, An electrochemical study of enzymatic oligonucleotide digestion, Bioelectrochem. Bioenerg. 63 (2004) 307–310. [9] Z. Zhou, J. Zhu, L. Zhang, Y. Du, S. Dong, E. Wang, G-quadruplex-based fluorescent assay of S1 nuclease activity and K+, Anal. Chem. 85 (2013) 2431– 2435. [10] Y. He, B. Jiao, H. Tang, Interaction of single-stranded DNA with graphene oxide: fluorescence study and its application for S1 nuclease detection, RSC Adv. 4 (2014) 18294–18300. [11] Z.D. Liu, P.P. Hu, H.X. Zhao, Y.F. Li, C.Z. Huang, Carbon nanotube–DNA hybrid used for activity monitoring and inhibitor screening of nuclease, Anal. Chim. Acta 706 (2011) 171–175. [12] Y. Tang, F. Feng, F. He, S. Wang, Y. Li, D. Zhu, Direct visualization of enzymatic cleavage and oxidative damage by hydroxyl radicals of single-stranded DNA with a cationic polythiophene derivative, J. Am. Chem. Soc. 128 (2006) 14972– 14976. [13] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362. [14] J. Lee, J. Park, H.H. Lee, H.I. Kim, W.J. Kim, DNA-templated silver nanoclusters as label-free, sensitive detection probes for potassium ion and nitric oxide, J. Mater. Chem. B 2 (2014) 2616–2621. [15] M. Zhang, S.M. Guo, Y.R. Li, P. Zuo, B.C. Ye, A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase, Chem. Commun. 48 (2012) 5488–5490 (Camb.). [16] M. Zhang, Y.Q. Liu, C.Y. Yu, B.C. Yin, B.C. Ye, Multiplexed detection of microRNAs by tuning DNA-scaffolded silver nanoclusters, Analyst 138 (2013) 4812–4817. [17] X. Xia, Y. Long, J. Wang, Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose, Anal. Chim. Acta 772 (2013) 81–86. [18] Y. Qian, Y. Zhang, L. Lu, Y. Cai, A label-free DNA-templated silver nanocluster probe for fluorescence on–off detection of endonuclease activity and inhibition, Biosens. Bioelectron. 51 (2014) 408–412. [19] K. Zhang, K. Wang, M. Xie, X. Zhu, L. Xu, R. Yang, B. Huang, X. Zhu, DNAtemplated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase, Biosens. Bioelectron. 52 (2014) 124– 128. [20] D. Schultz, S.M. Copp, N. Markesevic, K. Gardner, S.S. Oemrawsingh, D. Bouwmeester, E. Gwinn, Dual-color nanoscale assemblies of structurally stable, few-atom silver clusters, as reported by fluorescence resonance energy transfer, ACS Nano 7 (2013) 9798–9807. [21] L. Zhang, J. Zhu, Z. Zhou, S. Guo, J. Li, S. Dong, E. Wang, A new approach to light up DNA/Ag nanocluster-based beacons for bioanalysis, Chem. Sci. 4 (2013) 4004–4010. [22] A. Desireddy, B.E. Conn, J. Guo, B. Yoon, R.N. Barnett, B.M. Monahan, K. Kirschbaum, W.P. Griffith, R.L. Whetten, U. Landman, Ultrastable silver nanoparticles, Nature 501 (2013) 399–402. [23] J.T. Petty, J. Zheng, N.V. Hud, R.M. Dickson, DNA-templated Ag nanocluster formation, J. Am. Chem. Soc. 126 (2004) 5207–5212. [24] D. Schultz, K. Gardner, S.S. Oemrawsingh, N. Markesevic, K. Olsson, M. Debord, D. Bouwmeester, E. Gwinn, Evidence for rod-shaped DNA-stabilized silver nanocluster emitters, Adv. Mater. 25 (2013) 2797–2803. [25] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold nanoclusters, J. Am. Chem. Soc. 131 (2009) 888–889. [26] J. Zheng, R.M. Dickson, Individual water-soluble dendrimer-encapsulated silver nanodot fluorescence, J. Am. Chem. Soc. 124 (2002) 13982–13983. [27] J. Zheng, C. Zhang, R.M. Dickson, Highly fluorescent, water-soluble, sizetunable gold quantum dots, Phys. Rev. Lett. 93 (2004) 077402. [28] J. Liu, DNA-stabilized, fluorescent, metal nanoclusters for biosensor development, Trends Anal. Chem. 58 (2014) 99–111. [29] R. Hu, Y.R. Liu, R.M. Kong, M.J. Donovan, X.B. Zhang, W. Tan, G.L. Shen, R.Q. Yu, Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label free nuclease enzyme detection, Biosens. Bioelectron. 42 (2013) 31–35.
38
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
[30] Z. Qing, X. He, T. Qing, K. Wang, H. Shi, D. He, Z. Zou, L. Yan, F. Xu, X. Ye, Z. Mao, Poly(thymine)-templated fluorescent copper nanoparticles for ultrasensitive label-free nuclease assay and its inhibitors screening, Anal. Chem. 85 (2013) 12138–12143. [31] C.M. Ritchie, K.R. Johnsen, J.R. Kiser, Y. Antoku, R.M. Dickson, J.T. Petty, Ag nanocluster formation using a cytosine oligonucleotide template, J. Phys. Chem. C 111 (2007) 175–181.
[32] Y. Zhang, Y. Cai, Z. Qi, L. Lu, Y. Qian, DNA-templated silver nanoclusters for fluorescence turn-on assay of acetylcholinesterase activity, Anal. Chem. 85 (2013) 8455–8461. [33] M. Wang, X. Gu, G. Zhang, D. Zhang, D. Zhu, Continuous colorimetric assay for acetylcholinesterase and inhibitor screening with gold nanoparticles, Langmuir 25 (2009) 2504–2507.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Label-free fluorometric detection of S1 nuclease activity by using polycytosine oligonucleotide-templated silver nanoclusters Lihui Wang, Keke Ma, Yaodong Zhang ⇑ Key Laboratory of Applied Surface and Colloid Chemistry of Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
a r t i c l e
i n f o
Article history: Received 22 July 2014 Received in revised form 9 September 2014 Accepted 17 September 2014 Available online 28 September 2014 Keywords: S1 nuclease Silver nanoclusters Fluorometry Enzyme activity Enzyme inhibitor
a b s t r a c t S1 nuclease has an important function in DNA transcription, replication, recombination, and repair. A label-free fluorescent method for the detection of S1 nuclease activity has been developed using polycytosine oligonucleotide-templated silver nanoclusters (dC12–Ag NCs). In this assay, dC12 can function as both the template for the stabilization of Ag NCs and the substrate of the S1 nuclease. Fluorescent Ag NCs could be effectively formed using dC12 as the template without S1 nuclease. In the presence of S1 nuclease, dC12 is degraded to mono- or oligonucleotide fragments, thereby resulting in a reduction in fluorescence. S1 nuclease with an activity as low as 5 108 U ll1 (signal/noise = 3) can be determined with a linear range of 5 107 to 1 103 U ll1. The promising application of the proposed method in S1 nuclease inhibitor screening has been demonstrated using pyrophosphate as the model inhibitor. Furthermore, the S1 nuclease concentrations in RPMI 1640 cell medium were validated. The developed method for S1 nuclease is sensitive and facile because its operation does not require any complicated DNA labeling or laborious fluorescent dye synthesis. Ó 2014 Elsevier Inc. All rights reserved.
S1 nuclease hydrolyzes single-stranded DNA (ssDNA)1 or RNA into 50 -phosphomononucleotide and 50 -phosphooligonucleotide. This enzyme has been widely used in various applications such as in the determination of nucleic acid structure, the quantitation of nucleic acid hybridization, mapping mutations, detection of gaps in duplex DNA, and studying the interactions of DNA with various intercalating agents [1–3]. S1 nuclease has been particularly used to probe the disruption of the DNA structure by numerous carcinogens and antimitotic drugs [4]. Therefore, detecting S1 nuclease activity and screening for its potential inhibitors is substantially important. Numerous traditional methods have been used to measure the nuclease activity [5]. Although these methods are generally accurate, their routine use in laboratory is restricted because of their laborious and complicated conjugated chemistries. Recently, a number of colorimetric [6], electrochemical [7,8], and fluorescent [3,9,10] assays for S1 nuclease detection have been developed. In ⇑ Corresponding author at: Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China. Fax: +86 29 81530727. E-mail address: [email protected] (Y. Zhang). 1 Abbreviations used: ssDNA, single-stranded DNA; NC, nanocluster; Cu, copper; NP, nanoparticle; dC12, polycytosine oligonucleotide; Ag, silver; UV–Vis, ultraviolet– visible; PB, phosphate buffer. http://dx.doi.org/10.1016/j.ab.2014.09.011 0003-2697/Ó 2014 Elsevier Inc. All rights reserved.
addition, advanced materials, such as gold nanoparticles [6], carbon nanotubes [11], perylene derivatives [3], distyrylanthracene [10], and cationic polythiophene derivative [12], have been used for the construction of novel assay strategies for S1 nuclease. However, these strategies have specific drawbacks such as low detection sensitivity, expensive materials, and time-consuming assay procedures. Therefore, developing a highly sensitive and facile method for detecting S1 nuclease activity and screening for its potential inhibitors is substantially important. Noble metal nanoclusters (NCs) have been used in various applications, such as bioimaging, biosensing, and specific protein detection, because of their ultrafine size, nontoxicity, and good biocompatibility [13–21]. NCs can be routinely prepared in aqueous solutions with a number of thiolated or polymeric templates, including thiolated compounds [22], DNAs [23,24], proteins [25], and dendrimers [26,27]. DNA is a particularly interesting ligand for preparing fluorescent metal NCs and developing biosensors [28]. For the detection of S1 nuclease activity, two interesting DNA-templated NC methods, namely the fluorometric assay based on double-strand [29] or single-strand [30] DNA-templated copper (Cu) NCs/NPs (nanoparticles), have been recently developed. However, the relatively low quantum yield of Cu NCs/NPs (e.g., a value of 0.068 for T30-templated Cu NCs/NPs) [30] indicates that the detection sensitivity of these methods could be further improved.
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
In this study, polycytosine oligonucleotide (dC12) was used as both the S1 nuclease substrate and template for the formation of highly fluorescent dC12–Ag (silver) NCs. S1 nuclease degrades dC12 to nucleotide fragments, thereby resulting in a reduction in fluorescence. Thus, a label-free and highly sensitive method for S1 nuclease activity assay was developed. Materials and methods Materials and apparatus The S1 nuclease was provided by TaKaRa Biotechnology (Dalian, China). Oligonucleotides with dC12 were synthesized and purified by Sangon Biotechnology (Shanghai, China). The oligonucleotide stock solutions (200 lM) were prepared with deionized water and kept frozen at 20 °C. The RPMI 1640 cell medium was also purchased from Sangon Biotechnology. All other chemicals were of analytical grade and used without further treatment. Deionized water was prepared using a Millipore Milli-Q water purification system (18.2 MO). The ultraviolet–visible (UV–Vis) and fluorescence spectra of DNA–Ag NCs were obtained using a PerkinElmer Lambda 35 spectrometer and a PerkinElmer LS 55 spectrometer, respectively. Mass spectra were acquired in negative ion mode with 2.5-kV needle and 40-V cone voltages. Determination of S1 nuclease and inhibitory detection In total, 100 ll of the mixtures containing 20 lM dC12 and various concentrations of S1 nuclease ranging from 0.0 U ll1 to 5 102 U ll1 was incubated in an enzyme reaction buffer (30 mM CH3COONa, 100 mM NaNO3, and 1 mM Zn(NO3)2, pH 4.6) at 37 °C for 0.5 h in the PCR (polymerase chain reaction) tube. After enzyme digestion, the mixture was heated at 95 °C for 10 min to terminate the cleavage reaction. The solutions of sodium phosphate buffer (PB; 5 mM, pH 7.0, 92 ll) and AgNO3 (2 mM, 6 ll) were then separately introduced and further incubated in an ice bath for 15 min. Lastly, freshly prepared NaBH4 (5 mM, 2.5 ll) by dissolving NaBH4 powder in deionized water was added to induce fluorescent DNA–Ag NC formation. After being kept in the dark for 2 h at room temperature, the mixtures were diluted with PB for subsequent fluorescence measurements. The 10-fold amount (1 102 U/ll) of EcoRI, acid and alkaline phosphatase, ExoIII, and Escherichia coli ligase in the mixture was used to evaluate the specificity of the fluorometric assay to the S1 nuclease. The inhibition experiments were the same as for the above procedure except for the 15-min preferential incubation of the inhibitor with the S1 nuclease before the addition of dC12.
35
Results and discussion Detection mechanism of the method Scheme 1 illustrates the principle of the Ag NC-based fluorescent sensing system for S1 nuclease. The assay mechanism was based on function of dC12 as both the S1 nuclease substrate and template for the stabilization of Ag NCs. The oligonucleotide dC12 was selected as substrate because the dC12-templated silver clusters exhibit excellent fluorescence properties and have been well characterized by Ritchie and coworkers [31] and successfully used in bioanalysis [32]. In the absence of S1 nuclease, the strong fluorescent dC12–Ag NCs could be formed through the reduction of Ag+ by NaBH4. However, dC12 could be digested into mono- and/ or small fragments by the S1 nuclease, which results in the failed formation of Ag NCs because of the lack of a suitable template. Thus, S1 nuclease activity can be successfully determined through the change in the fluorescence of dC12–Ag NCs. Characteristics of Ag NCs and feasibility of the method The dC12–Ag NCs were synthesized according to the procedures in the literature [31,32]. To confirm the formation of the fluorescent dC12–Ag NCs, both UV–Vis and fluorescence spectra were analyzed. The absorbance spectra (Fig. 1, curve a) show two dominant peaks at maximum absorbance wavelengths of 443
Scheme 1. Schematic diagram of S1 nuclease activity assay based on dC12–Ag NCs.
Dynamic detection of S1 nuclease A series of 100-ll reaction mixtures containing a fixed concentration of 20 lM dC12 and 5 103 U ll1 S1 nuclease were incubated at 37 °C for 0, 1, 2, 3, 4, 5, 7, 10, 15, 20, and 30 min. After incubation for a predetermined period, the following experiments were performed according to the aforementioned procedure. Detection of S1 nuclease in complex samples To test the practicality of the proposed strategy, the target from the complex samples was analyzed according to the methods presented in the literature [30]. Different amounts of S1 nuclease were spiked into RPMI 1640 cell medium, in which 1 mM zinc nitrate was added to protect the enzyme activity. The S1 nuclease was subsequently detected via the proposed method, and the recovery value was determined.
Fig.1. UV–Vis (a) and fluorescence spectra of dC12–Ag NC probe: excitation curve (b), emission curve (c), incubation with inactivated S1 nuclease (d), and incubation with activated S1 nuclease (e). Maximum wavelengths of excitation and emission are 555 and 611 nm, respectively.
36
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
and 550 nm. The strongest fluorescence emission peak at 611 nm (curve c) was obtained on excitation at 555 nm (curve b). The oligonucleotide dC12 is bound with several silver atoms, as demonstrated in electrospray ionization mass spectra (see Fig. S1 in the online supplementary material). These results are similar to those reported in the literature [31] and indicate the formation of dC12–Ag NCs. To verify the feasibility of the designed assay strategy, the enzymatic cleavage reaction of S1 nuclease was investigated. dC12 with a concentration of 20 lM was incubated with 5 102 U ll1 S1 nuclease and with the same amount of inactivated S1 nuclease. After cleavage reaction at 37 °C for 30 min and subsequent heating at 95 °C for 10 min to terminate the reaction, the PB, AgNO3, and NaBH4 were added to induce fluorescent Ag NC formation. The resulting reaction solutions were then diluted and analyzed via fluorescence measurements. Fig. 1 shows that when dC12 was incubated with inactivated S1 nuclease and heated at 95 °C for 10 min to be denatured, the fluorescence intensity of dC12–Ag NCs (curve d) was almost the same as that without S1 nuclease (curve c). By contrast, the fluorescence intensity of the prepared dC12–Ag NCs significantly decreased (curve e) in the presence of active S1 nuclease. Therefore, the fluorescence decreased because of dC12 digestion by the S1 nuclease. The dynamic cleavage reaction of dC12 by the S1 nuclease was subsequently investigated. At cleavage times between 0 and 30 min, the fluorescence intensity at 611 nm of the obtained dC12–Ag NCs gradually decreased as a function of time (Fig. 2). The fluorescence rapidly decreased during the first 5 min and remained almost constant after 20 min, thereby demonstrating that the detection system can quickly respond to the S1 nuclease. Assay of nuclease activity To evaluate the assay performance, various amounts of S1 nuclease were added into the dC12 solutions and incubated for 0.5 h. The mixtures were then heated to 95 °C for 10 min to terminate the cleavage reaction and were used to prepare dC12–Ag NCs. Fig. 3 shows the obtained fluorescence spectra of the prepared dC12–Ag NCs. The fluorescence intensity at 611 nm decreased as a function of S1 nuclease concentration. The inset in Fig. 3 illustrates the linear relationship (R2 = 0.9966) between the fluorescence intensity ratio (F0/F) of DNA–Ag NCs at 611 nm and the S1 nuclease concentration ranging from 5 107 to 1 103
Fig.3. Fluorescence spectra of detection system with different S1 nuclease concentrations. Curves from a to k refer to 0.0, 5.0 108, 5.0 107, 5.0 106, 5.0 105, 2.5 104, 5.0 104, 1.0 103, 2.5 103, 5.0 103, and 5.0 102 U ll1, respectively. The inset shows the linear relationship of fluorescence intensity ratios (F0/F) at 611 nm versus S1 nuclease concentrations ranging from 5.0 107 to 1.0 103 U ll1.
U ll1, where F0 and F are the fluorescence intensities of the sensor solution in the absence and presence of the S1 nuclease, respectively. The detection limit was calculated to be 5 108 U ll1 according to the rule of 3 times the standard deviation over the blank response. The assay specificity was also tested by examining the interference of other common proteins or enzymes on the assay for S1 nuclease. As described in Materials and Methods, EcoRI, acid and alkaline phosphatase, ExoIII, and E. coli ligase were studied. Only S1 nuclease remarkably reduced the fluorescence intensities of the proteins or enzymes at the same conditions, and the values are almost equal to the maximum fluorescence emission intensity even though their concentrations were 10 times higher than that of the S1 nuclease (see Fig. S2 in supplementary material). These results indicate that the proposed method has a high selectivity toward nuclease. Inhibition assay of S1 nuclease Several studies have proven that the disruption of the S1 nuclease function is essential for many biological processes. Thus, the pharmacological inhibition of this enzyme provides a broad spectrum of therapeutic applications. In this study, we demonstrated the capacity of the developed method for detecting the inhibitors of the S1 nuclease by using pyrophosphate as the model inhibitor [11]. Fig. 4 shows that the relative activity of the S1 nuclease decreased with increasing pyrophosphate concentrations, and the concentration that caused a 50% fluorescence recovery is approximately 0.21 mM. The IC50 value was different from those reported before [1,11]. However, this is understandable given that the IC50 values usually depend on the measurement conditions such as method for determination, enzyme concentration, and pH [33]. These results demonstrate that the developed method has potential applications in screening for S1 nuclease-targeted drugs, which is important for antibiotics and anticancer therapeutics. Detection of S1 nuclease activity in complex samples
Fig.2. Fluorescence spectra of dC12–Ag NCs in the presence of S1 nuclease incubated for different periods. Inset: Time-dependent fluorescent changes at 611 nm. The S1 nuclease concentration is 1 103 U ll1.
To test the practicality of the proposed strategy, we chose the RPMI 1640 cell medium, into which five different S1 nuclease
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
37
References
Fig.4. Inhibitory effects of pyrophosphate on S1 nuclease.
Table 1 Measurement of S1 nuclease activity in RPMI 1640 cell medium. Added (103 U ll1)
Detected (103 U ll1)
Recovery (%)
RSD (%, n = 3)
0.050 0.10 0.50 5.0 10.0
0.047 0.097 0.49 4.8 9.5
94 97 98 96 95
6.7 5.4 7.9 5.2 8.1
Note. RSD, relative standard deviation.
concentrations (Table1) were spiked as the complex fluids. Table 1 shows that the obtained recoveries and reproducibility based on our strategy were desirable. These results indicate that the proposed method has considerable potential in practical applications for S1 nuclease detection with excellent accuracy and reliability. Conclusions We have provided a novel ultrasensitive and label-free fluorometric method for monitoring the cleavage of ssDNA by S1 nuclease and screening of enzyme inhibitors. The method depends on the function of dC12 as both the S1 nuclease substrate and Ag NC template. The significant decrease in the DNA–Ag NC emission could be used to monitor the DNA cleavage by the S1 nuclease. The detection limit is 5 108 U ll1, which is better than those obtained from previously reported methods [30]. The developed method not only is facile because its operation does not require any complex labeling of DNA or sophisticated experimental techniques but also offers a convenient strategy for a homogeneous and rapid detection with high sensitivity. Acknowledgments This work was supported by the National Natural Science Foundation of China (21275097) and the Fundamental Research Fund for the Central Universities (GK201303001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2014.09.011.
[1] R.C. Wiegand, G.N. Godson, C.M. Radding, Specificity of the S1 nuclease from Aspergillus oryzae, J. Biol. Chem. 250 (1975) 8848–8855. [2] N.A. Desai, V. Shankar, Single-strand-specific nucleases, FEMS Microbiol. Rev. 26 (2003) 457–491. [3] X. Yang, F. Pu, J. Ren, X. Qu, DNA-templated ensemble for label-free and realtime fluorescence turn-on detection of enzymatic/oxidative cleavage of singlestranded DNA, Chem. Commun. 47 (2011) 8133–8135 (Camb.). [4] J.L. Butour, A.M. Mazard, C. Vieussens, N.P. Johnson, Kinetic studies of the hydrolysis of platinum–DNA complexes by nuclease S1, Chem. Biol. Interact. 73 (1990) 195–205. [5] B. Mouratou, S. Rouyre, S. Pauillac, J.L. Guesdon, Development of nonradioactive microtiter plate assays for nuclease activity, Anal. Biochem. 309 (2002) 40–47. [6] R. Cao, B. Li, Y. Zhang, Z. Zhang, Naked-eye sensitive detection of nuclease activity using positively-charged gold nanoparticles as colorimetric probes, Chem. Commun. 47 (2011) 12301–12303 (Camb.). [7] J. Ding, W. Qin, Potentiometric sensing of nuclease activities and oxidative damage of single-stranded DNA using a polycation-sensitive membrane electrode, Biosens. Bioelectron. 47 (2013) 559–565. [8] S.C. Hillier, C.G. Frost, A.T. Jenkins, H.T. Braven, R.W. Keay, S.E. Flower, J.M. Clarkson, An electrochemical study of enzymatic oligonucleotide digestion, Bioelectrochem. Bioenerg. 63 (2004) 307–310. [9] Z. Zhou, J. Zhu, L. Zhang, Y. Du, S. Dong, E. Wang, G-quadruplex-based fluorescent assay of S1 nuclease activity and K+, Anal. Chem. 85 (2013) 2431– 2435. [10] Y. He, B. Jiao, H. Tang, Interaction of single-stranded DNA with graphene oxide: fluorescence study and its application for S1 nuclease detection, RSC Adv. 4 (2014) 18294–18300. [11] Z.D. Liu, P.P. Hu, H.X. Zhao, Y.F. Li, C.Z. Huang, Carbon nanotube–DNA hybrid used for activity monitoring and inhibitor screening of nuclease, Anal. Chim. Acta 706 (2011) 171–175. [12] Y. Tang, F. Feng, F. He, S. Wang, Y. Li, D. Zhu, Direct visualization of enzymatic cleavage and oxidative damage by hydroxyl radicals of single-stranded DNA with a cationic polythiophene derivative, J. Am. Chem. Soc. 128 (2006) 14972– 14976. [13] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362. [14] J. Lee, J. Park, H.H. Lee, H.I. Kim, W.J. Kim, DNA-templated silver nanoclusters as label-free, sensitive detection probes for potassium ion and nitric oxide, J. Mater. Chem. B 2 (2014) 2616–2621. [15] M. Zhang, S.M. Guo, Y.R. Li, P. Zuo, B.C. Ye, A label-free fluorescent molecular beacon based on DNA-templated silver nanoclusters for detection of adenosine and adenosine deaminase, Chem. Commun. 48 (2012) 5488–5490 (Camb.). [16] M. Zhang, Y.Q. Liu, C.Y. Yu, B.C. Yin, B.C. Ye, Multiplexed detection of microRNAs by tuning DNA-scaffolded silver nanoclusters, Analyst 138 (2013) 4812–4817. [17] X. Xia, Y. Long, J. Wang, Glucose oxidase-functionalized fluorescent gold nanoclusters as probes for glucose, Anal. Chim. Acta 772 (2013) 81–86. [18] Y. Qian, Y. Zhang, L. Lu, Y. Cai, A label-free DNA-templated silver nanocluster probe for fluorescence on–off detection of endonuclease activity and inhibition, Biosens. Bioelectron. 51 (2014) 408–412. [19] K. Zhang, K. Wang, M. Xie, X. Zhu, L. Xu, R. Yang, B. Huang, X. Zhu, DNAtemplated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase, Biosens. Bioelectron. 52 (2014) 124– 128. [20] D. Schultz, S.M. Copp, N. Markesevic, K. Gardner, S.S. Oemrawsingh, D. Bouwmeester, E. Gwinn, Dual-color nanoscale assemblies of structurally stable, few-atom silver clusters, as reported by fluorescence resonance energy transfer, ACS Nano 7 (2013) 9798–9807. [21] L. Zhang, J. Zhu, Z. Zhou, S. Guo, J. Li, S. Dong, E. Wang, A new approach to light up DNA/Ag nanocluster-based beacons for bioanalysis, Chem. Sci. 4 (2013) 4004–4010. [22] A. Desireddy, B.E. Conn, J. Guo, B. Yoon, R.N. Barnett, B.M. Monahan, K. Kirschbaum, W.P. Griffith, R.L. Whetten, U. Landman, Ultrastable silver nanoparticles, Nature 501 (2013) 399–402. [23] J.T. Petty, J. Zheng, N.V. Hud, R.M. Dickson, DNA-templated Ag nanocluster formation, J. Am. Chem. Soc. 126 (2004) 5207–5212. [24] D. Schultz, K. Gardner, S.S. Oemrawsingh, N. Markesevic, K. Olsson, M. Debord, D. Bouwmeester, E. Gwinn, Evidence for rod-shaped DNA-stabilized silver nanocluster emitters, Adv. Mater. 25 (2013) 2797–2803. [25] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold nanoclusters, J. Am. Chem. Soc. 131 (2009) 888–889. [26] J. Zheng, R.M. Dickson, Individual water-soluble dendrimer-encapsulated silver nanodot fluorescence, J. Am. Chem. Soc. 124 (2002) 13982–13983. [27] J. Zheng, C. Zhang, R.M. Dickson, Highly fluorescent, water-soluble, sizetunable gold quantum dots, Phys. Rev. Lett. 93 (2004) 077402. [28] J. Liu, DNA-stabilized, fluorescent, metal nanoclusters for biosensor development, Trends Anal. Chem. 58 (2014) 99–111. [29] R. Hu, Y.R. Liu, R.M. Kong, M.J. Donovan, X.B. Zhang, W. Tan, G.L. Shen, R.Q. Yu, Double-strand DNA-templated formation of copper nanoparticles as fluorescent probe for label free nuclease enzyme detection, Biosens. Bioelectron. 42 (2013) 31–35.
38
Fluorometric detection of S1 nuclease activity / L. Wang et al. / Anal. Biochem. 468 (2015) 34–38
[30] Z. Qing, X. He, T. Qing, K. Wang, H. Shi, D. He, Z. Zou, L. Yan, F. Xu, X. Ye, Z. Mao, Poly(thymine)-templated fluorescent copper nanoparticles for ultrasensitive label-free nuclease assay and its inhibitors screening, Anal. Chem. 85 (2013) 12138–12143. [31] C.M. Ritchie, K.R. Johnsen, J.R. Kiser, Y. Antoku, R.M. Dickson, J.T. Petty, Ag nanocluster formation using a cytosine oligonucleotide template, J. Phys. Chem. C 111 (2007) 175–181.
[32] Y. Zhang, Y. Cai, Z. Qi, L. Lu, Y. Qian, DNA-templated silver nanoclusters for fluorescence turn-on assay of acetylcholinesterase activity, Anal. Chem. 85 (2013) 8455–8461. [33] M. Wang, X. Gu, G. Zhang, D. Zhang, D. Zhu, Continuous colorimetric assay for acetylcholinesterase and inhibitor screening with gold nanoparticles, Langmuir 25 (2009) 2504–2507.
