Peptide-Mediated Energy Transfer between an Anionic Water-Soluble

16 Apr 2009 - Peptide-Mediated Energy Transfer between an. Anionic Water-Soluble Conjugated Polymer and. Texas Red Labeled DNA for Protease and...
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Anal. Chem. 2009, 81, 3731–3737

Peptide-Mediated Energy Transfer between an Anionic Water-Soluble Conjugated Polymer and Texas Red Labeled DNA for Protease and Nuclease Activity Study Yong Zhang, Yanyan Wang, and Bin Liu* Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576 We report for the first time that peptide could serve as a medium to bring an anionic conjugated polymer and a dye-labeled DNA into close proximity for energy transfer. By taking advantage of the fluorescein (Fl)-labeled peptide-mediated energy transfer between poly(9,9-bis(4′sulfonatobutyl)fluorene-alt-1,4-phenylene) sodium salt (PFP-SO3Na) and Texas red (TR)-labeled single-stranded DNA (ssDNA), we develop a homogeneous assay for detection and monitoring of protease and nuclease activity in one solution using peptide/DNA complexes as the substrate. The enzymes as a proof of concept are trypsin (protease) and S1 (nuclease), respectively. In the absence of enzyme, multistep fluorescence energy transfer occurs from PFP-SO3Na to Fl and TR and from Fl to TR, and the TR emission dominates the solution fluorescence. In the presence of trypsin, the peptide is cleaved into fragments; the relatively weak electrostatic interaction between the small peptide fragments and the polymer fails to bring the TR-ssDNA and the polymer into close proximity for energy transfer. There is a significant decrease in TR emission and an increase in PFP-SO3Na emission, and the solution fluorescence appears blue. When S1 nuclease is used to cleave TR-ssDNA, a significant decrease in TR emission and an obvious increase in Fl emission are found, and the solution fluorescence appears green. The developed assay is ideal for the detection of chemical and biological molecules with DNA or protein cleaving activities. The analysis and detection of enzymes is of high importance for the screening of noxious toxins and pathologies and for the development of efficient therapeutics. In particular, assays for proteases and nucleases are in great demand, due to the special functions of these enzymes in many important biological processes, such as protein catabolism, cell regulation and signaling, and DNA replication and repair.1-3 Traditional methods to monitor nuclease activity are based on gel electrophoresis, high-perfor* To whom correspondence should be addressed. E-mail: [email protected]. (1) Guarise, C.; Pasquato, L.; Filippis, V. D.; Scrimin, P. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 3978–3982. (2) Linn, S. M.; Lloyd, R. S.; Roberts, R. J. Nuclease, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1993. 10.1021/ac802488m CCC: $40.75  2009 American Chemical Society Published on Web 04/16/2009

mance liquid chromatography (HPLC), and enzyme-linked immunosorbent assays (ELISA).4-6 Standard assays for protease are based on radioisotopes or on fluorogenic substrates.7 These methods are either discontinuous, time-consuming, or require specific instruments. Many of these limitations have now being addressed by the development of novel detection strategies, among which the colorimetric- and fluorescence-based homogeneous assays have been widely studied.8-12 Most colorimetric assays for protease or nuclease take advantage of the aggregationor deaggregation-induced absorption or color change of gold nanoparticles upon analyte hydrolysis.1,8,9 These methods are convenient to use, but the sensitivity is often in the micromolar to submicromolar range. Although the fluorescence-based methods could provide higher detection sensitivity than the colorimetric methods, these assays generally use doubly labeled molecular beacons or peptides to serve as the substrates, which are expensive and difficult to synthesize.10–13 It remains a challenge to develop simple, rapid, and sensitive enzyme assays. Conjugated polymers (CPs) offer a unique platform for the development of highly sensitive fluorescence-based sensors for chemical and biological targets.14-24 The polymer backbone coor(3) Reich, E.; Rifkin, D. B.; Shaw, E. Proteases and Biological Control; Cold Spring Harbor Laboratory Press: Plainview, NY, 1975. (4) McLaughlin, L. W.; Benseler, F.; Graeser, E.; Piel, N.; Scholtissek, S. Biochemistry 1987, 26, 7238–7245. (5) Alves, J.; Ruter, T.; Geiger, R.; Fliess, A.; Maass, G.; Pingoud, A. Biochemistry 1989, 28, 2678–2684. (6) Jeltsche, A.; Fritz, A.; Alves, J.; Wolfes, H.; Pingoud, A. Anal. Biochem. 1993, 213, 234–240. (7) Reymond, J. L. Enzyme Assays: High-Throughput Screening, Genetic Selection, and Fingerprinting; John Wiley, 2006. (8) Ghadiali, J. E.; Stevens, M. M. Adv. Mater. [Online early access]. DOI: 10.1002/adma.200703158. Published Online: June 2, 2008. http://www3. interscience.wiley.com/journal/119814794/abstract?CRETRY)1)0. (9) Xu, X.; Han, M. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2007, 46, 3468– 3470. (10) Li, J. W. J.; Geyer, R.; Tan, W. H. Nucleic Acids Res. 2000, 28, e52. (11) Sun, H. Y.; Panicker, R. C.; Yao, S. Q. Biopolymers 2007, 88, 141–149. (12) Li, J. W. J.; Chu, Y. Z.; Lee, B. Y. H.; Xie, X. L. S. Nucleic Acids Res. 2008, 36, e36. (13) Medintz, I. L.; Clapp, A. R.; Brunel, F. M.; Tiefenbrunn, T.; Uyeda, H. T.; Chang, E. L.; Deschamps, J. R.; Dawson, P. E.; Mattoussi, H. Nat. Mater. 2006, 5, 581–589. (14) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954–10957. (15) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467–4476. (16) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942–1943. (17) Wang, Y. S.; Liu, B. Anal. Chem. 2007, 79, 7214–7220.

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dinates the action of a large number of absorbing units which allow efficient intramolecular and intermolecular energy transfer.25,26 Due to the delocalized electronic structure of CPs, the excitations can easily migrate to low-energy sites over a long distance. This property has been widely used to amplify the fluorescence signal of dye-labeled probes for DNA, RNA, and protein detection.14–19 Similarly, two types of CPE fluorescence-based methods have been developed for protease and nuclease activity study.27-29 One is based on the fluorescence recovery of an anionic CP which has been prequenched by a quencher-labeled peptide. The presence of a protease catalyzes the hydrolysis of the peptide backbone, leading to fluorescence recovery of the prequenched polymer emission.27,28 The other is based on fluorescence resonance energy transfer (FRET) between a cationic polyfluorene and dyelabeled oligonucleotide. The presence of an endonuclease cleaves the oligonucleotide, and the decrease in polymer-sensitized dye emission is used to monitor the enzyme activity.29 Although many strategies have been reported to study the enzyme activity, up to now, almost all the reported methods are limited to study a single enzyme in one solution. High-throughput detection of enzymes and determination of their activity and kinetic parameters is of great importance in the development of novel pharmaceuticals. In addition, successful molecular biology research requires constant monitoring of chemical and biological reagents free from contamination of both nuclease and protease. Although many artificial substrates have been developed for the measurement of enzyme activity, there is almost no report that a single substrate complex could be used to monitor both protease and nuclease activities in one solution. In this paper, we take advantage of a peptide-mediated energy transfer between a sulfonated polyfluorene derivative and Texas red labeled single-stranded DNA (TR-ssDNA) to study the activity of a protease and a nuclease enzyme in one solution. Trypsin (protease) and S1 (nuclease) are selected as the representative enzymes. As a common reagent in cell biology, trypsin is one of the most important digestive enzymes in controlling pancreatic exocrine function, which can predominantly cleave peptide chains at the carboxyl side of the lysine and arginine (18) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore´, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548–1551. (19) He, F.; Tang, Y. L.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2005, 127, 12343–12346. (20) Peter, K.; Nillsson, R.; Ingana¨s, O. Nat. Mater. 2003, 2, 419–424. (21) 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. (22) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, A. S.; 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. (23) Peter, K.; Nilsson, R.; Rydberg, J.; Baltzer, L.; Ingana¨s, O. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10170–10174. (24) Lee, K.; Povlich, L. K.; Kim, J. Adv. Funct. Mater. 2007, 17, 2580–2587. (25) Thomas, S. W., III.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339– 1386. (26) Swager, T. M. Acc. Chem. Res. 1998, 31, 201–207. (27) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400–3405. (28) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505– 7510. (29) Feng, F. D.; Tang, Y. L.; He, F.; Yu, M. H.; Duan, X. R.; Wang, S.; Li, Y. L.; Zhu, D. B. Adv. Mater. 2007, 19, 3490–3495.

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amino acids.30 S1 nuclease is an endonuclease and a ssDNAspecific nuclease that has been widely used in removing singlestranded overhangs from DNA fragments to yield blunt ends or in mapping of RNA transcripts and in probing the structures of purified DNA.31 EXPERIMENTAL SECTION General Information. Peptide-1 (sequence Arg-Arg-Arg-ArgArg-Arg-Arg-Arg-Arg-Arg) and peptide-2 (sequence Arg-Arg-ArgArg-Arg-Arg-Arg-Arg-Arg-Arg-FITC) were purchased from the GenScript Corporation, U.S.A. and used as received. The oligonucleotide (TR-ssDNA, 5′-TR-ATCTTGACTATGTGGGTGCT3′) was purchased from Research Biolabs, Singapore and used as received. S1 nuclease was obtained from Promega Corporation, and trypsin was purchased from Sigma. The 10× phosphate buffer saline (PBS buffer, ultrapure grade) is a commercial product from first BASE. Water was purified using a Millipore filtration system. The photoluminescence spectra were collected using a PerkinElmer LS-55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 photomultiplier tube (PMT), using 90° angle detection for solution samples. A gradient HPLC was performed on Shimadzu LC-6A apparatus with a UV detector SPD6AV (set at 220 nm) using a reversed-phase Zorbax 300ExtendC18 column (4.6 mm × 150 mm, 5 µm, Aligent Technologies) to monitor the peptide cleavage. A solution of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B) was used as the mobile phase. A gradient of 95% A to 60% A over 60 min at a flow rate of 0.5 mL/min was used for analysis. PBS buffer (45.7 mM NaCl, 0.9 mM KCl, 3.3 mM phosphate buffer, pH ) 8.0) was used for peptide digestion, and 1× S1 buffer (50 mM NaOAc, 280 mM NaCl, 4.5 mM ZnSO4, pH ) 4.5) was used for TR-ssDNA digestion. PFP-SO3Na was synthesized following the procedure in the literature.32 Synthesis of Poly(9,9-bis(4′-sulfonatobutyl)fluorene-coalt-1,4-phenylene) Sodium Salt (PFP-SO3Na). To the mixture of 2,7-dibromo-9,9-bis(4′-sulfonatobutyl)fluorene disodium (576.3 mg, 0.9 mmol), 1,4-phenylenebisboronic acid (148.3 mg, 0.9 mmol) and Pd(OAc)2 (10 mg) were mixed together in a mixture of DMF (30 mL) and aqueous Na2CO3 solution (0.2 M, 40 mL). After the mixture was degassed for 15 min, the reaction was vigorously stirred at 90 °C overnight under the argon atmosphere. After the reaction was cooled down to room temperature, the mixture was poured into acetone. The resulting precipitate was collected and dissolved in deionized water and was purified using a dialysis membrane with a cutoff molecular weight of 14 000 Da. The obtained solution was freeze-dried in vacuum to obtain the polymer of PFP-SO3Na (340 mg, 68%). 1 H NMR (300 MHz, DMSO-d6): δ 7.8-7.4 (m, 10H), 2.3 (br, 4H), 2.1 (br, 4H), 1.4 (br, 4H), 0.6 (br, 4H). 13C NMR (75 MHz, DMSO-d6): δ 151.7, 139.7, 139.1, 129.5, 127.6, 126.3, 120.9, 55.3, 51.5, 31.1, 25.4, 23.5. Energy Transfer between PFP-SO3Na and Peptide-2. To each 3 mL cuvette containing a mixture of [PFP-SO3Na] ) 1 × 10-7 M and [peptide-2] ) 5 × 10-9 M in 50 mM phosphate (30) Ionescu, R. E.; Cosnier, S.; Marks, R. S. Anal. Chem. 2006, 78, 6327– 6331. (31) Panayotatos, N.; Wells, R. D. Nature (London) 1981, 289, 466–470. (32) Huang, F.; Wang, X. H.; Wang, D. L.; Yang, W.; Cao, Y. Polymer 2005, 46, 12010–12015.

buffer, different amounts of peptide-1 with [peptide-1] varying from 1.5 × 10-8 M to 6 × 10-8 M were added. The mixture was gently shaken at room temperature before the fluorescence measurement. The excitation wavelength was 370 nm, and the fluorescence was collected in the range of 390-650 nm. The amount of peptide-1 required for maximum fluorescein (Fl) emission was identified based on the fluorescence intensity change at the Fl emission maximum (∼510 nm), and the highest Fl emission was observed at [peptide-1] ) 4.5 × 10-8 M upon excitation of the polymer at 370 nm. Peptide-Mediated Energy Transfer between PFP-SO3Na and ssDNA-TR. To each 3 mL cuvette containing [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, and [peptide-2] ) 5 × 10-9 M, different amounts of ssDNA-TR with [ssDNA-TR] ranging from 1.0 × 10-9 to 6.0 × 10-9 M were added dropwise. The solution fluorescence spectra were monitored upon excitation of PFP-SO3Na at 370 nm. In a separate experiment, the same amount of ssDNA-TR was added to solutions containing [PFP-SO3Na] ) 1 × 10-7 M and [peptide-1] ) 5 × 10-8 M to study the effect of Fl on the energy transfer between PFPSO3Na and ssDNA-TR. In these experiments, the excitation wavelength was 370 nm and the TR emission was monitored in the 550-750 nm range. The optimized concentrations of PFPSO3Na, peptide-1, peptide-2, and ssDNA-TR that give the highest TR signal are used for protease and nuclease study. Assay of Protease for Peptide Cleaving. Peptide cleaving solutions (50 µL) containing 5 × 10-5 M peptide ([peptide-1]/ [peptide-2] ) 9:1) and different amounts of trpysin were incubated at 37 °C in the digestion buffer solution (PBS buffer, pH ) 8.0). After a specific incubation time, 3 µL of the cleaved peptide solution was transferred into 3 mL of PBS buffer containing PFP-SO3Na (1 × 10-7 M) and TR-ssDNA (2 × 10-9 M). The fluorescence spectra were measured at room temperature under excitation at 370 nm. To check the peptide cleavage, 10 µL of solution taken from the mixture containing 5 × 10-5 M peptide-1 upon 60 min of digestion with 10 nM trypsin was injected into the HPLC. A solution of 0.1% TFA in water (A) and 0.1% TFA in acetonitrile (B) was used as the mobile phase. A gradient of 95% A to 60% A over 60 min at a flow rate of 0.5 mL/min was used for analysis. Assay of Nuclease for DNA Cleaving. A DNA cleaving solution with a total volume of 50 µL containing 5 × 10-6 M of TR-ssDNA and different amounts of S1 was incubated at 37 °C in 1× S1 buffer solution. After a specific incubation time, 1.2 µL of the cleaved ssDNA solution was taken out and transferred into a solution containing PFP-SO3Na (1 × 10-7 M) and peptide mixture (5 × 10-8 M, [peptide-1]/[peptide-2] ) 9:1) in 3 mL of PBS buffer (50 mM, pH ) 7.4). The fluorescence spectra were measured at room temperature under excitation at 370 nm. RESULTS AND DISCUSSION The chemical structures of PFP-SO3Na, Fl, and TR are shown in Scheme 1. The peptide and DNA sequences are also shown in Scheme 1. The strategy for monitoring trypsin and S1 nuclease activities is illustrated in Scheme 2. PFP-SO3Na has two negative charges on each repeat unit. It has bright blue fluorescence with the maximum absorption and emission wavelength at 370 and 415 nm, respectively. Both peptide-1 and peptide-2 have

Scheme 1. Chemical Structures of PFP-SO3Na, Fluorescein (Fl), and Texas Red (TR), and the Sequences of TR-ssDNA, Peptide-1, and Peptide-2

Scheme 2. Schematic Illustration of the Assay for Protease and Nuclease Activity Studya

a The scheme does not represent the scale and number of each component.

10 arginine units, and peptide-2 is labeled with Fl. Arginine has an isoelectric point of around 11, which indicates that each peptide has 10 positive charges at pH ) 7.4. The 20-base ssDNA is labeled with TR at its 5′-terminus. Fl has an absorption maximum at 488 nm and an emission maximum at 518 nm. TR has an absorption maximum at 589 nm and an emission maximum at 615 nm. There is an overlap between the emission of PFP-SO3Na and the absorption of Fl and TR, which indicates that FRET from PFP-SO3Na to Fl or TR is possible (Figure S1 in the Supporting Information). In addition, a good spectral overlap between Fl and TR also exists, which favors FRET from Fl to TR. With the use of the spectral data (shown in Supporting Information Figure S1) and the quantum yields for the polymer (70%) and Fl (80%) in buffer at pH ) 7.4, the Fo¨rster distance (R0)23 was calculated to be 37.5, 34.4, and 43.2 Å and for polymer/Fl, polymer/TR, and Fl/TR pairs, respectively, assuming an orientation factor (κ2) of 2/3. Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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The assay for enzyme activity study is shown in Scheme 2. One starts with a solution containing PFP-SO3Na, peptide-1, peptide-2, and TR-ssDNA. Both peptides serve as the bridge to bring PFP-SO3Na and TR-ssDNA into close proximity for energy transfer from the polymer to TR and from Fl to TR. The solution fluorescence is monitored by excitation of the polymer at 370 nm. Before enzyme digestion, TR emission (red) dominates the solution fluorescence. When the peptides are digested by trypsin into fragments, the relatively weak electrostatic interaction between peptide fragments and PFPSO3Na should prevent TR from coming into close proximity of PFP-SO3Na. Under this circumstance, emission from TR will decrease and that from PFP-SO3Na will increase. The polymer emission (blue) will dominate the solution fluorescence. On the other hand, if TR-ssDNA is cleaved by S1 nuclease, the relatively weak electrostatic interaction between TR-ssDNA fragments and the positively charged PFP-SO3Na/peptide complexes will cause TR to be separated from PFP-SO3Na and Fl. In this case, the TR emission intensity will decrease and the emission intensity for Fl and PFP-SO3Na will increase, which will lead to green fluorescence in solution. The cleavage of peptide or DNA by trypsin or S1 nuclease could lead to obvious fluorescence spectra changes, which allows monitoring of both protease and nuclease activity in the same solution. We started with optimization of the conditions for energy transfer between PFP-SO3Na and peptide-2. In a mixture of [PFPSO3Na] ) 1 × 10-7 M and [peptide-2] ) 1 × 10-8 M in 50 mM buffer, where the charge ratio (positive charges for peptide to negative charges for polymer) is 1:2, excitation of the polymer did not yield obvious Fl emission. Meanwhile, direct excitation of Fl at 490 nm revealed severe quenching (>95%) of the Fl emission as compared to that in the absence of the polymer (Figure S2 in the Supporting Information). This phenomenon is similar to our previous report that acceptor (C*) fluorescence quenching within cationic CP/ssDNA-C* complexes led to very low polymer-sensitized C* emission.33 This problem could be solved by introducing label-free probes of the same sequence to reduce the Fl-Fl interaction within the complexes.33 As a consequence, different amounts of peptide-1 were added to optimize the signal output of Fl upon excitation of PFP-SO3Na at 370 nm. The emission spectra for solutions containing 1 × 10-7 M PFP-SO3Na and 5 × 10-9 M peptide-2, upon addition of different amounts of peptide-1, are shown in Figure S3 in the Supporting Information. The highest polymer-sensitized Fl emission signal is observed for the solution containing 1 × 10-7 M PFP-SO3Na, 4.5 × 10-8 M peptide-1, and 5 × 10-9 M peptide-2. Under this condition, the charge ratio (±) is 2.5 in solution. The positively charged PFP-SO3Na/peptide complexes could be used to further complex with TR-ssDNA. Upon addition of TR-ssDNA with concentrations ranging from 1 × 10-9 M to 6 × 10-9 M to the above solution, a small decrease in polymer emission intensity was observed, while the Fl emission intensity decreased more quickly (Figure S4 in the Supporting Information). Upon excitation of the polymer, the maximum TR intensity was observed at [TR-ssDNA] close to 2 × 10-9 M, where the Fl emission peak was weak. This observation indicates that both PFP-SO3Na and Fl could serve (33) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188–1196.

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Figure 1. Emission spectra of PFP-SO3Na, PFP-SO3Na/peptide1,2, PFP-SO3Na/peptide-1/TR-ssDNA, and PFP-SO3Na/peptide-1,2/ TR-ssDNA. [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide-2] ) 5 × 10-9 M, [TR-ssDNA] ) 2 × 10-9 M. The excitation wavelength was 370 nm.

as energy donors for TR-ssDNA. To demonstrate the effect of Fl on energy transfer between PFP-SO3Na and TR-ssDNA, the emission spectrum of a solution containing 1 × 10-7 M PFPSO3Na, 5 × 10-8 M peptide-1, and 2 × 10-9 M TR-ssDNA is also collected, and the result is shown in Figure 1. The TR emission from solutions containing the polymer and peptide-1/2 is about 20% higher than that with only the polymer and peptide1, which further confirms that both PFP-SO3Na and Fl contribute to the TR emission upon excitation of the polymer at 370 nm. It is also important to note that although peptide-1,2 served as the bridge for energy transfer between PFP-SO3Na and TR-ssDNA, complex formation between PFP-SO3Na and peptide-1 also quenched the polymer fluorescence. Nonetheless, as shown in Figure 1, three distinct emission spectra could be observed for solutions containing PFP-SO3Na, PFP-SO3Na/ peptide-1,2, or PFP-SO3Na/peptide-1,2/TR-ssDNA. This makes the complex of PFP-SO3Na/peptide-1,2/TR-ssDNA a good substrate for both protease and nuclease assays. Figure 2a shows the fluorescence spectra change of PFPSO3Na/peptide-1,2/TR-ssDNA complexes as a function of trypsin digestion time. For trypsin digestion experiments, a solution containing 5 × 10-5 M peptide ([peptide-1]/[peptide2] ) 9:1) and 10 nM trypsin was incubated at 37 °C in the digestion buffer (50 mM PBS buffer, pH ) 8.0). After a specific incubation time, 3 µL of the solution was taken out and diluted with 3 mL of PBS buffer (50 mM, pH ) 7.4). After addition of PFP-SO3Na (1 × 10-7 M) and TR-ssDNA (2 × 10-9 M), the solution fluorescence was monitored. The initial emission of PFP-SO3Na/peptide1,2/TR-ssDNA showed intense red emission at 620 nm from TR and a weak green emission residue at 520 nm from Fl as well as blue emission with a moderate intensity at 420 nm from PFP-SO3Na. With trypsin digestion, the emission intensity of TR at 620 nm gradually decreased over the incubating time from 0 to 24 min. Meanwhile, the blue emission from PFP-SO3Na at 420 nm showed a gradual increase over the full incubating time. This indicates that the strength of electrostatic interaction between PFP-SO3Na and peptides decreases over digestion time due to the formation of small peptide fragments, which increases the distance between PFP-

Figure 2. (a) Emission spectra of PFP-SO3Na/peptide-1,2/ TR-ssDNA as a function of trypsin digestion upon excitation at 370 nm. [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide2] ) 5 × 10-9 M, [TR-ssDNA] ) 2 × 10-9 M, [trypsin] ) 10 nM. The trypsin digestion was conducted in PBS buffer (50 mM, pH ) 8.0). (b) Time-dependent TR fluorescence intensity as a function of trypsin concentration for solutions containing [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide-2] ) 5 × 10-9 M, and [TR-ssDNA] ) 2 × 10-9 M. The excitation wavelength was 370 nm. The data are based on the average of three independent experiments with error bars indicated.

SO3Na and TR, leading to inefficient FRET between them. When the peptide was further digested to 60 min, both fluorescence from Fl and TR almost disappeared and there was an obvious increase in PFP-SO3Na intensity. The solution showed blue fluorescence upon excitation of the polymer at 370 nm (Figure S5 in the Supporting Information). Reversedphase HPLC was also used to monitor the peptide digestion. Comparison of the HPLC spectra for peptide-1 before and after digestion (Figure S6 in the Supporting Information for 5 × 10-5 M peptide-1 upon 60 min of digestion with 10 nM trypsin) reveals new peaks at short elution times after peptide digestion, which correspond to small peptide fragments. The integrated area of the peak for peptide-1 after digestion is about 30% as compared to that before digestion. This result indicates that it is unnecessary to cleave all the peptide into small fragments in order to induce a significant decrease in TR emission.

To demonstrate the application of PFP-SO3Na/peptide-1,2/ TR-ssDNA complex in monitoring the time-dependent protease activity, the fluorescence intensity of TR at 620 nm was studied as a function of protease concentration. Figure 2b shows the response of TR intensity to peptide digestion reaction with trypsin concentrations varying from 0 to 30 nM. At each trypsin concentration, there is a decrease in TR intensity with increased incubation time, which is followed by a plateau. After incubation of the solution for 4 min, the TR intensity decreases ∼80% for solutions containing 15 nM trypsin, while only ∼20% decrease is observed in the presence of 10 nM trypsin. This observation indicates that increasing the trypsin concentration gives rise to a higher cleavage reaction rate and less time is required to complete the digestion process. For 10 nM trypsin, almost 70% decrease in TR intensity is observed after incubation of the complex for 12 min. The PFP-SO3Na/peptide-1,2/TR-ssDNA complex was also used to monitor S1 nuclease activity. The changes in fluorescence spectra of the complex as the function of S1 nuclease digestion time are shown in Figure 3a. The incubation was conducted at 37 °C in 1× S1 digestion buffer (50 µL) containing 5 × 10-6 M TR-ssDNA and 6 U/mL S1 nuclease. At a specific incubation time, 1.2 µL of the solution was taken out and diluted with 3 mL of PBS buffer (50 mM, pH ) 7.4) containing PFPSO3Na (1 × 10-7 M), peptide-1 (4.5 × 10-8 M), and peptide-2 (5 × 10-9 M). The solution fluorescence was monitored upon excitation of the polymer at 370 nm. As shown in Figure 3a, the TR emission intensity shows a gradual decrease with increasing incubation time from 0 to 70 min. The decrease in TR intensity indicates the reduced interaction between PFP-SO3Na/peptide complex and TR after S1 nuclease digestion. Along with the decrease in TR emission intensity, the Fl emission intensity gradually increases with the incubation time. The increased Fl intensity can be attributed to less efficient energy transfer from Fl to TR due to DNA digestion. For the nuclease assay, the time-dependent changes in TR emission upon incubation of 2 × 10-9 M TR-ssDNA with S1 nuclease at [S1 nuclease] ) 0-12 U/mL are shown in Figure 3b. The TR emission intensity decreases with increased incubation time at each S1 nuclease concentration. In addition, the fluorescence intensity decreases more rapidly for the cleavage reaction at a higher concentration of S1 nuclease with an increased reaction rate. Kinetic investigation of S1-digested reaction using the PFPSO3Na/peptide-1,2/TR-ssDNA complex as the substrate has been further studied. From 0 to 2 nM TR-ssDNA was added to each solution containing 1 × 10-7 M PFP-SO3Na], 4.5 × 10-8 M [peptide-1], and 5 × 10-9 M [peptide-2], and the TR emission intensity (PL) at 620 nm was monitored upon excitation of PFPSO3Na at 370 nm. As shown in Figure S7 in the Supporting Information, the ratio of PL/PL0 (PL0 is the TR intensity for 2 nM TR-ssDNA in the substrate solution upon excitation at 370 nm) is then plotted against [TR-ssDNA] to yield a calibration curve, which allows us to calculate the amount of TR-ssDNA remaining in the substrate solution at any specific digestion time by measuring the PL/PL0. From Figure 3b and Supporting Information Figure S7, one can easily derive the plots for [TR-ssDNA] remaining in solution with respect to the digestion time in the presence of different [S1], and the result is Analytical Chemistry, Vol. 81, No. 10, May 15, 2009

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Figure 3. (a) Emission spectra of PFP-SO3Na/peptide-1,2/ TR-ssDNA as a function of S1 nuclease digestion upon excitation at 370 nm. [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide-2] ) 5 × 10-9 M, [TR-ssDNA] ) 2 × 10-9 M, [S1] ) 6 U/mL. The nuclease digestion was conducted in 1× S1 digestion buffer (pH ) 4.5). (b) The time-dependent TR fluorescence intensity as a function of digestion time with varying S1 nuclease concentration for solutions containing [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide-2] ) 5 × 10-9 M, and [TR-ssDNA] ) 2 × 10-9 M. The excitation wavelength was 370 nm. The data are based on the average of three independent experiments with error bars indicated.

shown as Figure S8 in the Supporting Information. The slopes of the plots at early time were calculated to afford the initial rate of S1-digested reaction (v0) for the four different concentrations of S1 investigated, and the results are shown in Figure 4a. A linear plot of v0 as a function of [S1] from 0 to 12 U/mL indicates that the digestion is kinetically controlled by S1 nuclease. On the basis of the method reported by Liu and Schanze,34 the suitability of peptide-mediated S1 digestion assay has been investigated by Michaelis-Menten analysis.35 By taking the natural logarithm of [TR-ssDNA], Supporting Information Figure S8 could be transformed into Figure 4b. The kinetic parameter (Vmax/Km)36 is derived from the slope of each plot. (34) Liu, Y.; Schanze, K. S. Anal. Chem. 2008, 80, 8605–8612. (35) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry; Worth Publishers: New York, 1993. (36) Vmax is the maximum rate of the enzyme-catalyzed reaction at saturation substrate concentration; Km is the Michaelis-Menten constant, which is the substrate concentration where the rate of the enzyme-catalyzed reaction is half Vmax.

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Figure 4. Enzyme kinetics measurement: (a) the linear relation between the initial rate of S1 digestion reaction (v0) and nuclease S1 concentrations; (b) natural logarithm of [TR-ssDNA] as a function of digestion time for various [S1]. Inset: the kinetic parameters (Vmax/ Km) as a function of nuclease S1 concentration. 1 U of nuclease S1 ) 7.8 × 10-14 mol. The solution contains [PFP-SO3Na] ) 1 × 10-7 M, [peptide-1] ) 4.5 × 10-8 M, [peptide-2] ) 5 × 10-9 M, and [TR-ssDNA] ) 2 × 10-9 M for enzyme digestion.

As shown in Figure 4b, plotting the kinetic parameter versus [S1] yields a linear line. Considering that 1 U of S1 nuclease contains 7.8 × 10-14 mol, the specificity constant (kcat/Km)37 is calculated to be 1.12 × 106 M-1 s-1 from the slope of the plot. This constant is in same order of magnitude as that reported by Li et al. using molecular beacon as the substrate.10 These results demonstrate that it is possible to detect S1 nuclease activity using the PFP-SO3Na/peptide-1,2/TR-ssDNA complex as the substrate. In conclusion, we have demonstrated a new strategy for homogeneous detection of protease and nuclease using complexes of PFP-SO3Na, Fl-labeled peptide, and TR-labeled ssDNA. This method combines multistep fluorescence energy transfer from PFP-SO3Na to Fl and TR and from Fl to TR. Upon excitation of the polymer, the solution fluorescence is red when in the absence of enzyme, green when nuclease is present, and blue when protease is found. This assay allows qualitative study of protease and nuclease activity using the same substrate, which (37) kcat is the catalytic constant or turnover number.

opens new opportunities for high-throughput screening of enzymes. In addition, quantitative kinetic data could also be derived from the enzyme assay for S1 nuclease activity study. However, due to the large amount of peptide-1 presented in the substrate and that the peptide quenches the polymer fluorescence, this assay is not suitable for kinetic investigation of protease activities. Further development of substrates by selecting suitable polymer-dye pairs with minimized energy waste channels could eliminate the use of nonlabeled peptide, leading to more efficient assays that allow monitoring and quantitative analysis of several enzymes in one solution. The concept for multiple enzyme study with the same substrate could also be extended to other systems, such as fluorescence turn-on/off assays and cationic CP-based assays for different enzymes. This study opens new opportunities for highthroughput screening of enzymes and their inhibitors using a standard 96-well microliter plate.

ACKNOWLEDGMENT The authors are grateful to the National University of Singapore (NUS ARF R-279-000-197-112/133, R-279-000-233-123, R-279-000234-123) and Singapore Ministry of Education (R-279-000-255-122) for financial support. SUPPORTING INFORMATION AVAILABLE The PL spectra, the HPLC spectra for the digested peptide, and the calibration curve for TR fluorescence vs [TR-ssDNA]. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review November 24, 2008. Accepted March 26, 2009. AC802488M

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