Universal Platform for Sensitive and Label-Free Nuclease Assay

Dec 3, 2009 - By taking advantage of the large signal amplification through efficient fluorescence resonance energy transfer (FRET) from the conjugate...
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Universal Platform for Sensitive and Label-Free Nuclease Assay Based on Conjugated Polymer and DNA/Intercalating Dye Complex Fang Pu,† Dan Hu,† Jinsong Ren,*,† Shu Wang,‡ and Xiaogang Qu† †

State Key Laboratory of Rare Earth Resources Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China and ‡Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China Received September 10, 2009. Revised Manuscript Received November 15, 2009

By taking advantage of the large signal amplification through efficient fluorescence resonance energy transfer (FRET) from the conjugated polymer to the intercalating dye mediated by DNA, a new strategy for nuclease assay has been developed using conjugated polymer and DNA/intercalating dye complex. The discrimination of DNA before or after digestion by nuclease denotes the universal application of the approach, in which either dsDNA or ssDNA substrates could be used for detecting nuclease activity. This method can be extended to most nucleases by simply changing the substrate DNA. The present method is label-free, rapid, and highly sensitive, with a detection limit much better or at least comparable to previous reports. In addition, this assay is easy to implement for visual detection with the assistance of a UV transilluminator. We reason that a similar strategy can be adopted for the detection of other analytes.

Introduction DNA or RNA cleavage reactions catalyzed by nucleases such as restriction nucleases and nonspecific nucleases are essential in a variety of fields ranging from biotechnology to pharmacology, as well as in biological processes involving replication, recombination, DNA repair, molecular cloning, genotyping, and mapping.1-5 Recent attempts to attach restriction endonucleases to nanostructures have been undertaken due to the well-established ability of the endonucleases to cleave DNA at specific recognition sites.6-8 Traditional techniques, including gel electrophoresis, high-performance liquid chromatography (HPLC), electrochemical study, and enzymelinked immunosorbent assay (ELISA) have been established in assaying nuclease activities.9-13 These protocols share the drawbacks of being time-intensive, DNA-consuming, discontinuous, laborious, and usually requiring isotope labeling. Many of these limitations are now being addressed by the development of fluorescence assays based on fluorescence quenching or *To whom correspondence should be addressed. E-mail: [email protected]. (1) Desai, N. A.; Shankar, V. FEMS Microbiol. Rev. 2003, 26, 457–491. (2) Arber, W. Angew. Chem., Int. Ed. 1978, 17, 73–79. (3) Pingoud, A.; Jeltsch, A. Nucleic Acids Res. 2001, 29, 3705–3727. (4) Marti, T. M.; Fleck, O. Cell. Mol. Life Sci. 2004, 61, 336–354. (5) Grindley, N. D.; Whiteson, K. L.; Rice, P. A. Annu. Rev. Biochem. 2006, 75, 567–605. (6) Kanaras, A. G.; Wang, Z.; Hussain, I.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 67–70. (7) Song, G.; Chen, C.; Ren, J.; Qu, X. ACS Nano 2009, 3, 1183–1189. (8) Kanaras, A. G.; Wang, Z.; Brust, M.; Cosstick, R.; Bates, A. D. Small 2007, 3, 590–594. (9) Alves, J.; Ruter, T.; Geiger, R.; Fliess, A.; Maass, G.; Pingoud, A. Biochemistry 1989, 28, 2678–2684. (10) McLaughlin, L. W.; Benseler, F.; Graeser, E.; Piel, N.; Scholtissek, S. Biochemistry 1987, 26, 7238–7245. (11) Hillier, S. C.; Frost, C. G.; Jenkins, A. T.; Braven, H. T.; Keay, R. W.; Flower, S. E.; Clarkson, J. M. Bioelectrochemistry 2004, 63, 307–310. (12) Fujita, K.; Kanazawa, M.; Mukumoto, K.; Nojima, T.; Sato, S.; Kondo, H.; Waki, M.; Takenaka, S. Nucleic Acids Symp. Ser. 2006, 307–308. (13) Jeltsch, A.; Fritz, A.; Alves, J.; Wolfes, H.; Pingoud, A. Anal. Biochem. 1993, 213, 234–340. (14) Li, J. J.; Geyer, R.; Tan, W. Nucleic Acids Res. 2000, 28, E52. (15) Biggins, J. B.; Prudent, J. R.; Marshall, D. J.; Ruppen, M.; Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13537–13542.

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fluorescence resonance energy transfer (FRET).14-17 Although promising, these techniques are compromised by the requirement for double-labeled DNA probes, limited chemical stability, and interferences by external nonspecific events. Very recently, a gold nanoparticle (AuNP)-based application has been developed for assaying nuclease activity.8,18,19 For example, a colorimetric endonuclease-inhibition method has been reported based on polymeric aggregates of DNA-functionalized AuNP with DNA-duplex interconnects.19 And an alternative AuNP-FRET approach has been described for detection of single-stranded DNA (ssDNA) nuclease activity using fluorescent dye-labeled DNA.20 However, the need of functionalizing thiol (or dye)-modified oligonucleotide probes added to the complexity, cost, and overall assay speed and limited the effectiveness to such detection strategies. It is still desirable to develop sensitive, facile, and label-free strategies to assay nuclease activity. Recently, the use of conjugated polymers (CPs) as either chemical or biological sensing elements has received intense research interest. CPs contain a large number of absorbing and delocalized molecular units, and the transfer of excitation energy along the whole backbone of the CP to the energy/electron acceptor results in an amplification of the fluorescence signals.21 Therefore, CPs have been successfully employed in the detection (16) Yang, C. J.; Li, J. J.; Tan, W. Methods Mol. Biol. 2006, 335, 71–81. (17) Ma, C.; Tang, Z.; Wang, K.; Tan, W.; Yang, X.; Li, W.; Li, Z.; Lv, X. Anal. Biochem. 2007, 363, 294–296. (18) Zhao, W.; Lam, J. C.; Chiuman, W.; Brook, M. A.; Li, Y. Small 2008, 4, 810–816. (19) Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468– 3470. (20) Ray, P. C.; Fortner, A.; Darbha, G. K. J. Phys. Chem. B 2006, 110, 20745– 20748. (21) (a) Swager, T. M. Acc. Chem. Res. 1998, 31, 201–207. (b) Thomas, S. W., 3rd; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339–1386. (c) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537–2574. (d) Bunz, U. H. Chem. Rev. 2000, 100, 1605–1644. (e) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467– 4476. (f) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168–178. (g) Feng, F. D.; He, F.; An, L. L.; Wang, S.; Li, Y. H.; Zhu, D. B. Adv. Mater. 2008, 20, 2959–2964. (h) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater. Chem. 2005, 15, 2648–2656.

Published on Web 12/03/2009

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of various substances including DNA, RNA, protein, and metal ions22-28 and in the sensing of pH27a-c and temperature.27d Recently, we reported a colorimetric (or fluorescence) method to monitor nuclease activity using polythiophene derivatives (or cationic polyfluorene complex).29,30 However, these assays are limited by either their need of chromophore-labeled substrate or the fact that they are only for single-strand-specific nuclease detection with lower sensitivity. Herein, we demonstrate a new concept that overcomes these limitations by achieving a sensitive, reliable, and universal platform for label-free detection of nuclease activity based on CPs/DNA/ interacting dye system.

Experimental Section Materials and Measurement. All oligonucleotides were purchased from Sangon Biotechnology Inc. (Shanghai, China) and used without further purification. ssDNA with the sequence GGTTGGTGTGGTTGG (TBA) and its complementary sequence CCAACCACACCAACC (CTBA) were chosen for nonrestriction nucleases study. GTA TGA GAA TTC TCA TAC (DNA1) was chosen for restriction nucleases study. The DNA concentration was determined by measuring the absorbance at λ = 260 nm at high temperature (90 °C) by using a JASCO V-550 spectrophotometer, equipped with a temperature-controlled cuvette holder controlled by using a circulating bath. Genefinder was purchased from Bio-v Company (Xiamen, China). S1 nuclease was obtained from TaKaRa Biotechnology Co., Ltd. DNase I was purchased from Promega Corporation. EcoR I was purchased from New England Biolabs. The nucleases were put on ice before use. The water was purified using a Millipore filtration system. Fluorescence spectra were obtained with a JASCO FP6500 spectrophotometer (Jasco International Co., Ltd., Tokyo, (22) (a) Chen, L.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287–12292. (b) Kumaraswamy, S.; Bergstedt, T.; Shi, X.; Rininsland, F.; Kushon, S.; Xia, W.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511–7515. (c) Chemburu, S.; Corbitt, T. S.; Ista, L. K.; Ji, E.; Fulghum, J.; Lopez, G. P.; Ogawa, K.; Schanze, K. S.; Whitten, D. G. Langmuir 2008, 24, 11053–11062. (23) (a) Yang, C. Y. J.; Pinto, M.; Schanze, K.; Tan, W. H. Angew. Chem., Int. Ed. 2005, 44, 2572–2576. (b) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S. A. 2004, 101, 7505–7510. (c) Liu, Y.; Ogawa, K.; Schanze, K. S. Anal. Chem. 2008, 80, 150–158. (d) Liu, Y.; Schanze, K. S. Anal. Chem. 2009, 81, 231–239. (24) (a) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548–1551. (b) Dore, K.; Dubus, S.; Ho, H. A.; Levesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 4240– 4244. (c) Ho, H. A.; Bera-Aberem, M.; Leclerc, M. Chem.;Eur. J. 2005, 11, 1718– 1724. (d) Aberem, M. B.; Najari, A.; Ho, H. A.; Gravel, J. F.; Nobert, P.; Boudreau, D.; Leclerc, M. Adv. Mater. 2006, 18, 2703–2707. (25) (a) He, F.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2005, 127, 12343–12346. (b) Duan, X.; Li, Z.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154–4155. (c) Feng, F. D.; Wang, H. Z.; Han, L. L.; Wang, S. J. Am. Chem. Soc. 2008, 130, 11338–11343. (d) Feng, F. D.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. Angew. Chem., Int. Ed. 2007, 46, 7882–7886. (26) (a) Nilsson, K. P.; Inganas, O. Nat. Mater. 2003, 2, 419–424. (b) Nilsson, K. P.; Rydberg, J.; Baltzer, L.; Inganas, O. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10170–10174. (c) Sigurdson, C. J.; Nilsson, K. P.; Hornemann, S.; Manco, G.; Polymenidou, M.; Schwarz, P.; Leclerc, M.; Hammarstrom, P.; Wuthrich, K.; Aguzzi, A. Nat. Methods 2007, 4, 1023–1030. (d) Herland, A.; Nilsson, K. P.; Olsson, J. D.; Hammarstrom, P.; Konradsson, P.; Inganas, O. J. Am. Chem. Soc. 2005, 127, 2317– 2323. (27) (a) McQuade, D. T.; Hegedus, A. H.; Swager, T. M. J. Am. Chem. Soc. 2000, 122, 12389–12390. (b) Piletsky, S. A.; Panasyuk, T. L.; Piletskaya, E. V.; Elskaya, A. V.; Pringsheim, E.; Wolfbeis, O. S. Fresenius’ J. Anal. Chem. 2000, 366, 807–810. (c) Pringsheim, E.; Zimin, D.; Wolfbeis, O. S. Adv. Mater. 2001, 13, 819–822. (d) Kuroda, K.; Swager, T. M. Macromolecules 2004, 37, 716–724. (28) (a) Kim, I. B.; Erdogan, B.; Wilson, J. N.; Bunz, U. H. Chem.;Eur. J. 2004, 10, 6247–6254. (b) Phillips, R. L.; Miranda, O. R.; You, C. C.; Rotello, V. M.; Bunz, U. H. F. Angew. Chem., Int. Ed. 2008, 47, 2590–2594. (c) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H. F.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318–323. (29) Tang, Y.; Feng, F.; He, F.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2006, 128, 14972–14976. (30) (a) Feng, F.; Tang, Y.; He, F.; Yu, M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2007, 19, 3490–3495. (b) Feng, X.; Duan, X.; Liu, L.; Feng, F.; Wang, S.; Li, Y.; Zhu, D. Angew. Chem., Int. Ed. 2009, 48, 5316–5321.

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Article Japan). Fluorescence images were obtained with GDS8000 UV transilluminator. Assay of DNase I or EcoR I for dsDNA Cleavage. In the experiments, a solution with a total volume of 50 μL containing 5  10-6 M dsDNA and various amounts of DNase I or EcoR I in buffer (Tris-HCl 40 mM, MgSO4 10 mM, CaCl2 1 mM, pH 8.0 for DNase I; 100 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, and 0.025% Triton X-100, pH 7.5 for EcoR I) was incubated at 37 °C. After a specific incubation period, the cleavage reaction was stopped with 37 mM EDTA. Then 5 μL of the solution was drawn out and diluted with 400 μL of H2O. Poly[(9,9-bis (60 -N,N,Ntrimethylammonium)-hexyl]-fluorenylene phenylene dibromide] (PFP) ([PFP] = 2.0 μM in RUs (repeated units)) and Genefinder (0.01 ) were added, and the fluorescence spectra were measured with the excitation wavelength of 380 nm. Assay of S1 Nuclease for ssDNA Cleavage. A solution with a total volume of 50 μL containing 5  10-6 M TBA and various amounts of S1 nuclease in buffer (CH3COONa 30 mM, NaCl 280 mM, ZnSO4 1 mM, pH 4.6) was incubated at 37 °C. After a specific incubation time, 5 μL of the solution was drawn out and the cleavage reaction was stopped with 37 mM EDTA. Then the solution was diluted with 400 μL of H2O containing 6  10-8 M CTBA. PFP ([PFP] = 2.0 μM in RUs (repeated units)) and Genefinder (0.01 ) were added, and the fluorescence spectra were measured at room temperature with the excitation wavelength of 380 nm.

Results and Discussion Design of the Strategy for DNA Cleavage. Our new strategy for nuclease assay relies on the efficient energy transfer from the cationic conjugated polymer to the intercalating dye mediated by DNA, a phenomenon originally reported by Bazan and co-workers.31,32 As shown in Scheme 1, our system is composed of three elements: label-free DNA substrate, an intercalating dye, and water-soluble cationic polymer, PFP (structure shown in Figure 1A). The electrostatic interactions between the positively charged PFP and the negatively charged oligonucleotides will bring PFP and intercalators into close proximity, allowing for the FRET from PFP to the intercalating dye. Therefore, a large signal amplification (emission increase) of the intercalating dye and/or fluorescence quenching of the PFP is observed. In the presence of nuclease, the DNA substrate is cleaved into small fragments. In this case, the formation of the dsDNA structure required for dye intercalation would not take place and inefficient FRET would be observed. Thus, the cleavage of DNA by nuclease can be monitored by fluorescence spectra by observing PFP or intercalating dye emission change in aqueous solutions. DNA intercalating dyes such as ethidium bromide (EB), thiazole orange (TO), and YOYO (an oxazole yellow dimer) have been used as acceptors for label-free DNA detection. However, inefficient energy transfer was observed due to a nonoptional (orthogonal) orientation between the transition moment of the optically active polymer backbone and that of the intercalated dyes within the dsDNA structure.31,33 And in certain cases, the detection was limited to G-rich DNA only.32 To overcome these problems, we introduce an intercalating cyanine dye, Genefinder, as the energy acceptor instead. The emission maximum of PFP is centered at 420 nm, and the absorbance maximum of Genefinder is 495 nm (Figure S1, Supporting Information). The large spectra overlap in the 400-550 nm range between the emission spectrum (31) Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446– 5451. (32) Liu, B.; Bazan, G. C. Macromol. Rapid Commun. 2007, 28, 1804–1808. (33) Tian, N.; Xu, Q. H. Adv. Mater. 2007, 19, 1988–1991.

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Scheme 1. Schematic Illustration of the Strategy for Label-Free Nucleases Detection Using Conjugated Polymer PFP and Intercalating Dye Genefinder: (A) Cleavage of dsDNA by DNase I or EcoR I and (B) Cleavage of ssDNA by S1 Nuclease

Figure 1. (A) Molecular structure of PFP. (B) Fluorescence spectra of the solution containing ssDNA/Genefinder or dsDNA/ Genefinder in the presence of PFP, upon excitation at 380 nm. Direct excitation of ssDNA/Genefinder or dsDNA/Genefinder at 500 nm in the absence of PFP is also shown. [PFP] = 2  10-6 M, [ssDNA or dsDNA] = 1  10-7 M. (C) Fluorescence images of PFP, PFP/Genefinder, PFP/Genefinder/ssDNA, PFP/Genefinder/dsDNA, Genefinder, Genefinder/ssDNA, and Genefinder/ dsDNA from 1 to 7, respectively.

of PFP and the absorption spectrum of Genefinder met the requirement for efficient energy transfer between conjugated polymers and the DNA intercalator. The energy-transfer process would then result in the significant amplification of fluorescence signals. Figure 1B compares the fluorescence emission spectra of PFP ([PFP] = 2  10-6 M) and Genefinder (with an optimized amount of 0.01 for efficient FRET and selectivity) in the presence of DNA ([ssDNA] or [dsDNA] = 1  10-7 M). Upon the addition of dsDNA, the fluorescence emission of PFP was decreased when excited at 380 nm, whereas the emission of Genefinder was significantly amplified 9-fold in comparison with that by direct excitation of Genefinder at 500 nm as shown in Figure 1B. This large signal amplification clearly indicates an efficient FRET from PFP to Genefinder. Interestingly, nearly no FRET was observed upon excitation of PFP for the PFP/ssDNA/ Genefinder system. These features denote the universal application of the approach, in which either dsDNA or ssDNA substrates could be used for assaying nuclease activity (Scheme 1). Moreover, the fluorescence difference for DNA/Genefinder solution in the absence and presence of PFP is readily distinguishable by the naked eye under UV illumination with an excitation wavelength of 365 nm. As shown in Figure 1C, the tube containing Genefinder/dsDNA (or ssDNA) was dim in the absence of PFP. In contrast, bright emission was observed for the PFP/Genefinder/ dsDNA complex. The fluorescence images were consistent with 4542 DOI: 10.1021/la904173j

the fluorescence spectra, and efficient signal amplification by PFP could be easily visualized under a hand-held UV lamp. As demonstrated here, the PFP/dsDNA/intercalating dye system could serve as a sensitive and label-free platform for assaying nuclease activity in real time. Assay for the DNA Cleavage by DNase I. Nuclease detection using dsDNA as the substrate was first demonstrated. DNase I is a nonrestriction nuclease that degrades dsDNA, producing 30 -OH oligonucleotides, and is extensively used in probing genomic DNA, removing the DNA template after in vitro transcription and nick translation.34,35 As illustrated in Scheme 1A, before enzymatic digestion, the electrostatic interactions between PFP and dsDNA keep them in close proximity, allowing for efficient FRET from PFP to Genefinder. Therefore, fluorescence quenching of the PFP and emission increase of Genefinder are observed. After enzymatic digestion by DNase I, small fragments are generated and no dye intercalation occurs. Adding of PFP to these solutions does not induce any FRET. In this case, the increase of emission of PFP and the decrease of emission of Genefinder could be observed. Thus, the cleavage of dsDNA by DNase I can be monitored by fluorescence spectra by observing PFP and Genefinder emission changes in aqueous solution. In a typical experiment, solutions with a total volume of 50 μL containing 5  10-6 M dsDNA and various amounts of DNase I in buffer (Tris-HCl 40 mM, MgSO4 10 mM, CaCl2 1 mM) at pH 8.0 were incubated at 37 °C. After a specific incubation period, 5 μL of the solution was drawn out and diluted with 400 μL of H2O. PFP ([PFP] = 2.0 μM in RUs (repeated units)) and Genefinder (0.01 ) were added, and the fluorescence spectra were measured at room temperature. Upon addition of DNase I, the emission intensity of PFP at 420 nm gradually (34) Anderson, S. Nucleic Acids Res. 1981, 9, 3015–3027. (35) Kienzle, N.; Young, D.; Zehntner, S.; Bushell, G.; Sculley, T. B. BioTechniques 1996, 20, 612–616.

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increased, and that of Genefinder at 520 nm gradually decreased over the incubation time from 0 to 80 min (Figure S2A, Supporting Information). This shows that the interactions of PFP and dsDNA get weaker and weaker following DNA digestion and inefficient FRET occurs. The ratio of emission intensity at 420 and 520 nm (I420nm/ I520nm), which is associated with the population of cleaved DNA, was then studied as a function of incubating time. Figure 2A shows the time curves of the DNA digestion reactions with various concentrations of DNase I from 0.5  10-3 to 0.1 unit μL-1. It is found that the ratio values of I420nm/I520nm increased with the nuclease incubation and reached the plateau after 40 min, which indicated that the digestion of dsDNA was nearly completed. It is possible to detect the enzyme activity within ten minutes, which showed one of the advantages of quick response of the system. The reaction rate increases with increasing enzyme concentration and can be followed in real time. The initial digestion rate can be measured from the linear portion of the time curve, and can then be used to determine the enzyme activity in the cleavage reaction. Because the amount of DNA remaining in the solution at a certain reaction time was reduced with the increase of concentrations of nucleases, quantification of the nuclease activity was obtained from the plot of the ratio values of I420nm/I520nm versus nuclease concentrations. As can be seen in Figure 2B, the population of cleaved DNA was greatly dependent on enzyme concentration and a sigmoid working curve could be obtained. The detection range of DNase I in the current assay was shown to range from approximately 5  10-4 to 0.02 unit μL-1, which is comparable to the AuNP-based method.18 The unit of nuclease used here to measure the amount of nuclease is traditional activity unit defined by classic digestion experiments. As a novel method, it could define a unit that can correlate the fluorescent signals with the amount of enzymes. Fluorescent data were converted to substrate concentration by using the DNA-fluorescence calibration curve. Then the data of time-course set corresponding to enzyme concentration were converted to percent digestion by setting the negative control (no enzyme reaction) as 0% and the positive control (complete digestion) as 100%. Finally, percentage values were converted to number of base pairs by setting the number of base pairs found in the full-length substrate as 100%. Assuming that one enzyme binds to a DNA and catalyzes hydrolysis, the experiment could suggest the amount of nuclease needed to digest specific number of nucleotides per second. In addition to fluorescence analysis, the fluorescence difference for the digestion solution could be visualized using UV transilluminator. The gradual fluorescence changes of the solution from bright to dim were observed following the dsDNA digestion by DNase I from 0 to 60 min. The brightness did not decrease any more after 40 min, which indicated that the digestion of dsDNA was nearly completed (Figure 2C). Furthermore, the concentration dependent enzymatic cleavage could also allow for naked-eye detection. As seen clearly in Figure 2D, the fluorescence changed from bright to dim along with the increase of DNase I concentration. These results are consistent with the FRET results and strongly support that our fluorescence amplification strategy provides a convenient method for assaying nuclease activity. Assay for the DNA Cleavage by EcoR I. To address the generality of our approach, detection of enzyme activity of restriction nuclease was also studied. EcoR I, a type II restriction endonuclease,3 was used as a model system. Since the recognition sites of type II endonucleases are typically short, palindromic sequences of dsDNA, the substrate DNA1 was designed to form a self-complementary 18 base pair duplex DNA, in which the single Langmuir 2010, 26(6), 4540–4545

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Figure 2. (A) Ratio of emission at the wavelengths 420/520 nm (I420/I520) versus digestion time of dsDNA by DNase I with various nuclease concentrations. (B) I420/I520 versus nuclease concentrations at a fixed concentration of DNA. [PFP] = 2  10-6 M, [dsDNA] = 6  10-8 M. Digestion time was 1 h at 37 °C. Excitation wavelength was 380 nm. (C) Fluorescence images of the solutions containing PFP/dsDNA/Genefinder before and after digestion by DNase I. Sample 1: PFP/Genefinder. Samples 2-10: solutions digested by 4  10-3 unit μL-1 DNase I for 0, 5, 10, 15, 20, 30, 40, 50, and 60 min, respectively. (D) Fluorescence images of the concentration dependent enzymatic cleavage. Sample 1: PFP/ Genefinder. Samples 2-10: PFP/dsDNA/Genefinder with 0, 0.2, 0.5, 1, 2, 4, 8, 10, and 20 unit mL-1 DNase I, respectively.

restriction site of 50 -d (GAATTC)-30 was incorporated into the duplex region. The cleavage of the dsDNA1 was first analyzed by polyacrylamide gel electrophoresis (PAGE). As shown in Figure S3 in the Supporting Information, the dsDNA1 is cleaved by specific EcoR I nuclease. We then investigated the DNA cleavage by EcoR I with the established method. The reaction buffer contains 50 mM NaCl, 100 mM Tris-HCl, 10 mM MgCl2, and 0.025% Triton X-100 (pH 7.5). Because the two short fragments cleaved by EcoR I could still remain double-stranded at room temperature, allowing for the FRET from PFP to Genefinder through intercalation, so we carried out the detection at 37 °C (Scheme 1A). At this temperature, the products had enough energy to dissociate and the difference of fluorescence before and after digestion could then be detected. As shown in Figure 3A, similar to the case of DNA cleavage by the nonrestriction nuclease DNase I, the emission intensity of PFP at 420 nm increased and that of Genefinder at 520 nm decreased with the increase of EcoR I concentration. In addition, the photographs taken from the solution showed consistent fluorescence intensity DOI: 10.1021/la904173j

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Figure 3. (A) I420/I520 versus EcoR I concentrations at a fixed concentration of DNA. [PFP] = 2  10-6 M, [DNA] = 5  10-8 M. Digestion time was 5 h at 37 °C. (B) Fluorescence images of the concentration dependent enzymatic cleavage. Sample 1: PFP/Genefinder/DNA1. Samples 2-7: PFP/Genefinder/DNA1 with 5  10-4, 5  10-3, 0.02, 0.1, 1.0, and 2.0 unit μL-1 EcoR I, respectively.

change (Figure 3B). Note that the detection limit for the restriction enzyme in this work (1  10-3 unit μL-1) is comparable to that of a molecular beacon probe17 due to the amplification of fluorescence signals by PFP. Assay for the DNA Cleavage by S1 Nuclease. Our universal platform for label-free detection was further applied to single-strand-specific nucleases. Single-strand-specific nucleases are widespread multifunctional enzymes, which act selectively on single strand and single-stranded regions in double-stranded nucleic acids. They have shown extensive application as probes for the structural determination of nucleic acids. Intracellularly, they have been implicated in recombination, repair and replication, whereas extracellular enzymes have a role in nutrition.1 S1 nuclease is chosen as the model enzyme. As shown in Scheme 1B, the ssDNA substrate hybrids with its complementary sequence to form dsDNA structure in the absence of S1. The strong electrostatic interactions between the PFP and dsDNA can bring the PFP and intercalating dye into proximity, enabling efficient energy transfer to occur. However, when the ssDNA substrate is cleaved by S1 nuclease into small fragments, the formation of the dsDNA structure required for dye intercalation would not take place and inefficient FRET would be observed. Thus, the cleavage of ssDNA by S1 nuclease can be observed by fluorescence spectra changes of PFP and Genefinder. The assay begins with the hydrolization of ssDNA by various amounts of S1 nuclease in buffer solution (CH3COONa 30 mM, NaCl 280 mM, ZnSO4 1 mM) at pH 4.6. Then the complementary sequence was added and fluorescence spectra were measured with the excitation wavelength 380 nm in the presence of PFP and Genefinder. As expected, the emission intensity of the PFP at 420 nm gradually increased and that of Genefinder at 520 nm gradually decreased over the incubating time from 0 to 80 min. By measuring the ratio I420nm/I520nm, the time curves at a fixed concentration of ssDNA with various S1 nuclease concentrations were obtained (Figure 4A). A detection limit of 2.6  10-6 unit μL-1 was obtained from the sigmoid working curve (Figure 4B). In addition, the observed fluorescence images for S1 nuclease using a UV transilluminator were fully consistent with fluorescence 4544 DOI: 10.1021/la904173j

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Figure 4. (A) I420/I520 versus digestion time of ssDNA by S1 nuclease with various nuclease concentrations. (B) I420/I520 versus nuclease concentrations at a fixed concentration of DNA. [PFP] = 2  10-6 M, [DNA] = 6  10-8 M. (C) Fluorescence images of the solutions containing PFP/DNA/Genefinder before and after digestion by S1 nuclease. Sample 1: PFP/Genefinder. Sample 2: PFP/ Genefinder/ssDNA. Samples 3-8: the solution digested by 8  10-3 unit μL-1 S1 nuclease for 0, 5, 10, 20, 40, and 60 min, respectively. (D) Fluorescence images of the concentration dependent enzymatic cleavage. Sample 1: PFP/Genefinder. Samples 2-10: PFP/Genefinder/DNA with 0, 0.5, 1, 2, 3, 4, 5, 6, and 8 unit mL-1 S1 nuclease, respectively.

spectra. Fluorescence changed from bright to dim along with the increase of digesting time (Figure 4C) or S1 nuclease concentration (Figure 4D). Significantly, the detection limit for S1 in our work is at least 1 order of magnitude below the published data obtained by gel electrophoresis, chromatography, UV-based assay, and FRET methods and is comparable to the value of 2.8  10-6 unit μL-1 obtained by the fluorescein labeled DNA/cationic conjugated polymer system.30 The label-free and ratiometric sensing mode here offers additional advantages over previous reports.

Conclusion In conclusion, we have demonstrated a unique design of a universal platform for sensitive, facile, and label-free nuclease detection using a CPs/DNA/intercalating dye complex. The method relies on the large signal amplification through efficient FRET from the conjugated polymer to the intercalating dye mediated by DNA. Compared with previous studies where chromophore-labeled DNA substrate is needed or only singlestrand-specific nuclease could be detected with lower sensitivity, the assay presented here is simple in design and offers a convenient “mix-and-detect” protocol for homogeneous, ratiometric, and rapid detection with high sensitivity. The detection limit is much better or at least comparable to previous reports. In addition, this assay is easy to implement for visual detection under a hand-held UV lamp. More importantly, this approach avoids either CP modification or oligonucleotide labeling, which offers the advantages of simplicity and cost efficiency. Furthermore, the approach shows potential for high-throughput screening of enzyme inhibitors, and a similar strategy can also be adopted for the detection of other analytes, such as proteins and small molecules. We expect that this strategy may have important applications in a wide range of fields such as biology, biomedicine, process control, and nanotechnology. Langmuir 2010, 26(6), 4540–4545

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Acknowledgment. Financial support was provided by the National Natural Science Foundation of China, the Fund from CAS and Jilin Province. Supporting Information Available: Fluorescence spectrum of PFP and absorption spectrum of Genefinder; fluorescence

Langmuir 2010, 26(6), 4540–4545

Article

spectra of PFP/DNA/Genefinder with the change of nuclease digestion time; fluorescence spectra of PFP/DNA/Genefinder with various amounts of nucleases; electrophoresis analysis of cleavage of DNA-1 by EcoR I. This material is available free of charge via the Internet at http://pubs. acs.org.

DOI: 10.1021/la904173j

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