Conjugated Polyelectrolyte Amplified Thiazole Orange Emission for

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Conjugated Polyelectrolyte Amplified Thiazole Orange Emission for Label Free Sequence Specific DNA Detection with Single Nucleotide Polymorphism Selectivity Kai Li and Bin Liu* Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore We report a simple label-free method for sequence specific DNA detection using cationic conjugated polyelectrolyte (CCP) amplified thiazole orange (TO) emission as the signal. The discrimination of perfectly complementary DNA from a one-base mismatched sequence is accomplished by S1 nuclease digestion of the hybridized peptide nucleic acid (PNA)/DNA complexes. When the target DNA is complementary to the PNA probe, the DNA/ PNA complexes remain after digestion, which allows TO intercalation to give fluorescence. Addition of CCP to this solution leads to enhanced TO emission due to fluorescence resonance energy transfer (FRET) from the CCP to TO, and the solution fluorescence appears yellow. In the presence of even one base mismatched DNA or doublestranded DNA molecules, S1 nuclease can effectively digest the DNA sequences into small fragments and no dye intercalation occurs. Addition of CCP to these solutions does not induce any polymer fluorescence change, and the solution fluorescent color is blue. This method allows visual detection of target DNA with a detection limit of 5 µM, which provides good groundwork for the future exploration of real-time instrument free single nucleotide polymorphism (SNP) diagnosis. Single nucleotide polymorphisms (SNPs) represent a natural genetic variability found at high density in the human genome, as often as every few hundred to thousand base pairs in genomic DNA. When SNPs occur, both the structure and the function of the encoded protein are changed, often leading to harmful diseases. Convenient, economical, and real-time detection and analysis of SNPs is of high importance for identifying the diseasecausing genes for early disease diagnosis and treatment.1-5 * To whom correspondence should be addressed. E-mail: cheliub@ nus.edu.sg. (1) Halushka, M. K.; Fan, J. B.; Bentley, K.; Hsie, L.; Shen, N.; Weder, A.; Cooper, R.; Lipshutz, R.; Chakravarti, A. Nat. Genet. 1999, 22, 239–247. (2) (a) Rothberg, B. E. G. Nat. Biotechnol. 2001, 19, 209–211. (b) Wang, D. G.; Fan, J. B.; Siao, C. J.; Berno, A.; Young, P.; Sapolsky, R.; Ghandour, G.; Perkins, N.; Winchester, E.; Spencer, J.; Kruglyak, L.; Stein, L.; Hsie, L.; Topaloglou, T.; Hubbell, E.; Robinson, E.; Mittmann, M.; Morris, M. S.; Shen, N. P.; Kilburn, D.; Rioux, J.; Nusbaum, C.; Rozen, S.; Hudson, T. J.; Lipshutz, R.; Chee, M.; Lander, E. S. Science 1998, 280, 1077–1082. 10.1021/ac9003985 CCC: $40.75  2009 American Chemical Society Published on Web 04/16/2009

A number of genotyping methods used for SNP detection have been reported in the literature.6-15 Among these methods, peptide nucleic acid (PNA) based detection has shown great potentials for rapid and accurate detection of oligonucleotide mutations.15-18 One strategy is to use PNA beacons, which have been modified with a dye on one end and a quencher on the other side. In the absence of DNA targets, the beacon forms a closed loop where the quencher and the dye are in close proximity to quench the dye fluorescence. The presence of a complementary DNA sequence opens up the beacon and induces dye emission. The selectivity of PNA beacon based assay is often restricted when the difference in stability between perfectly matched and mismatched duplexes is not obvious enough to allow distinct diagnosis. Recently, S1 nuclease is introduced to PNA based DNA SNP detection as it is able to hydrolyze the mismatched DNA sequences while keeping the perfectly matched DNA sequence intact.The effectiveness of such detection strategies is often limited (3) Hacia, J. G.; Fan, J.-B.; Ryder, O.; Jin, L.; Edgemon, K.; Ghandour, G.; Mayer, R. A.; Sun, B.; Hsie, L.; Robbins, C. M.; Brody, L. C.; Wang, D.; Lander, E. S.; Lipshutz, R.; Fodor, S. P. A.; Collins, F. S. Nat. Genet. 1999, 22, 164–167. (4) Tsongalis, G. J.; Coleman, W. B. Clin. Chim. Acta 2006, 363, 127–137. (5) McCathy, J. J.; Hilfiker, R. Nat. Biotechnol. 2002, 18, 505–508. (6) (a) Xu, H.; Wu, H. P.; Huang, F.; Song, S. P.; Li, W. X.; Cao, Y.; Fan, C. H. Nucleic Acids Res. 2005, 33, e83. (b) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 34– 39. (c) Wang, Y. S.; Liu, B. Anal. Chem. 2007, 79, 7214–7220. (d) Duan, X. R.; Li, Z. P.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154–4155. (7) Wang, J.; Nielsen, P. E.; Jiang, M.; Cai, X. H.; Fernandes, J. R.; Grant, D. H.; Ozsoz, M.; Beglieter, A.; Mowat, M. Anal. Chem. 1997, 69, 5200–5202. (8) Komiyama, M.; Ye, S.; Liang, X.; Yamamoto, Y.; Tomita, T.; Zhou, J. M.; Aburatani, H. J. Am. Chem. Soc. 2003, 125, 3758–3762. (9) Wang, H.; Li, J.; Liu, H.; Liu, Q.; Mei, Q.; Wang, Y.; Zhu, J.; He, N.; Lu, Z. Nucleic Acid Res. 2002, 30, e61. (10) Svanvik, N.; Westman, G.; Wang, D. Y.; Kubista, M. Anal. Biochem. 2000, 281, 26–35. (11) Ross, P.; Hall, L.; Smirnov, I.; Haff, L. Nat. Biotechnol. 1998, 16, 1347– 1351. (12) Wei, F.; Chen, C.; Zhai, L.; Zhang, N.; Zhao, X. S. J. Am. Chem. Soc. 2005, 127, 5306–5307. (13) Livak, K. J. Genet. Anal.: Biomol. Eng. 1999, 14, 143–149. (14) Irizarry, K.; Kustanovich, V.; Li, C.; Brown, N.; Nelson, S.; Wong, W.; Lee, C. J. Nat. Genet. 2000, 26, 233–236. (15) Kuhn, H.; Demidov, V. V.; Coull, J. M.; Fiandaca, M. J.; Gildea, B. D.; Kamenetskii, M. D. F. J. Am. Chem. Soc. 2002, 124, 1097–1103. (16) Seitz, O. Angew. Chem., Int. Ed. 2000, 39, 3249–3252. (17) Ross, P. L.; Lee, K.; Belgrader, P. Anal. Chem. 1997, 69, 4197–4202. (18) Ren, B. Z.; Zhou, J. M.; Komiyama, M. Nucleic Acid Res. 2004, 32, e42.

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by the sensitivity of the fluorescent 19,20 reporter.19 In addition, a majority of PNA based DNA detection methods require singly or dually labeled probes, which add to the complexity and cost. It is highly desirable to develop strategies for label free sequence specific detection with high sensitivity and selectivity. Fluorescent intercalator based detection is one of the classical methods that has been widely used to monitor DNA hybridization. Intercalation dyes increase their emission efficiencies upon incorporation within the internally stacked bases of doublestranded DNA (dsDNA).21 Thiazole orange (TO), as a member of the family of asymmetric cyanines, has been widely used as a signal reporter for DNA diagnostics.22 The intercalator based dsDNA detection is simple and effective but lacks specificity since the intercalation dye can interact with almost any dsDNA molecule in solution to generate fluorescent signals. Recently, TO labeled PNA light up probes have been reported to show sequence specific DNA detection.23,24 However, the fluorescence signal from intercalated TO for PNA/DNA is relatively low due to the dramatic reduction in binding capability of TO to PNA/DNA as compared to that of dsDNA.25 One strategy to improve the sensitivity and selectivity of the intercalator based assays is to take advantage of fluorescence resonance energy transfer (FRET) from the donor molecule to the intercalation dye.26 In previous studies, we have shown that both cationic oligomers and CCPs could be used as energy donors to amplify the intercalation dye emission.6c,27 The signal amplification is due to the large absorption coefficient and the delocalized electronic structure of the donors, which allow efficient energy transfer to the signal reporter dye.28 By taking advantage of CCP sensitized TO emission and the specificity of PNA probe and S1 nuclease, in this contribution, we report a label-free DNA detection strategy with good sensitivity and SNP selectivity. With the assistance of a hand-held UV lamp, this strategy also allows nakedeye detection of DNA in solution. EXPERIMENTAL SECTION Materials. The HPLC-purified PNAd (Lys-Lys-OO-GCT ACT GAC C) and PNAt probes (TCC TTC TCC T-OO-Lys-Lys) were purchased from Panagene. The DNA oligonucleotides have the following sequences: 5′-TCC TTC TCC T-3′ (DNAt of the same sequence as PNAt), 3′-CGA TGA CTG G-5′ (DNAdc, (19) Ye, S.; Miyajima, Y.; Ohnishi, T.; Yamamoto, Y.; Komiyama, M. Anal. Biochem. 2007, 363, 300–302. (20) Demidov, V.; Frank-Kamenetskii, M. D.; Egholm, M.; Buchardt, O.; Nielsen, P. E. Nucleic Acids Res. 1993, 21, 2103–2107. (21) Lepecq, J. B.; Paoletti, C. J. Mol. Biol. 1967, 27, 87–106. (22) Seitz, O.; Bergmann, F.; Heindl, D. Angew. Chem., Int. Ed 2999, 38, 2203– 2206. (b) Zhu, H.; Clark, S. M.; Benson, S. C.; Rye, H. S.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1994, 66, 1941–1948. (c) Jarikote, D. V; Krebs, N.; Tannert, S.; Ro ¨der, B.; Seitz, O. Chem.sEur. J. 2007, 13, 300–310. (23) Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers 1998, 46, 39–51. (24) Svanvik, N.; Nygren, J.; Westman, G.; Kubista, M. J. Am. Chem. Soc. 2001, 123, 803–809. (25) Wittung, P.; Kim, S. K.; Buchardt, O.; Nielsen, P.; Norden, B. Nucleic Acids Res. 1994, 22, 5371–5377. (26) (a) Talavera, E. M.; Bermejo, R.; Crovetto, L.; Orte, A.; Alvarez-Pez, J. M. Appl. Spectrosc. 2003, 57, 208–215. (b) Algar, W. R.; Massey, M.; Krull, U. J. J. Fluores. 2006, 16, 555–567. (27) (a) Liu, B.; Dan, T. T. T.; Bazan, G. C. Adv. Funct. Mater. 2007, 17, 2432– 2438. (b) Liu, B.; Bazan, G. C. Macromol. Rapid Commun. 2007, 28, 1804– 1808. (28) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339– 1386.

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Scheme 1. Molecular Structures of PFP and TO and Sequences of PNA and DNAa

a The mismatched positions of DNAd1 and DNAt1 are shown in bold italic.

complementary to PNAd), 3′-AGG AAG AGG A-5′ (DNAtc, complementary to PNAt), 3′-CGA TTA CTG G-5′ (DNAd1, onebase mismatch to PNAd), 3′-AGG AAT AGG A-5′ (DNAt1, onebase mismatch to PNAt). All the HPLC-purified DNA oligonucleotides were obtained from Sigma-Aldrich and used as received. The stock solutions of PNA and DNA molecules were made in Milli-Q water. S1 nuclease and 10× S1 buffer (500 mM sodium acetate, 2.8 M sodium chloride, and 45 mM zinc sulfate, pH 4.5) were purchased from Promega. Milli-Q water was used to prepare all buffers. Thiazole orange (TO) was purchased from Aldrich and used without further purification. Poly{2,7[9,9-bis(6′-N,N,N-trimethylammoniumhexyl)] fluorene-co-1,4phenylene dibromide} (PFP) was synthesized according to the literature.29 The molecular weight and polydispersity of PFP were 25 000 and 2.1, respectively. The chemical structures of PFP and TO and the sequences of PNA and DNA are shown in Scheme 1. Instruments. The UV-vis absorption spectra were obtained from a Shimadzu UV-1700 spectrometer. Fluorescence was measured using a Perkin-Elmer LS 55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90° angle detection for solution samples. For energy transfer experiments, the excitation beam intensity has been automatically corrected with a correction file provided by the instrument supplier. Photographs of the polymer solution were taken using a Canon EOS 400D digital camera under a hand-held UV-lamp with λmax ) 365 nm. The hybridization and digestion experiments were conducted using an Eppendorf thermomixer. Hybridization Procedures. To prepare PNAd/DNAdc and PNAd/DNAd1 duplexes, the PNAd probe (30 µL, 200 µM) and DNAdc or DNAd1 sequence (60 µL, 100 µM) were mixed together in Milli-Q water (108 µL) with the addition of NaCl (2 µL, 500 mM). The PNA2t/DNAtc and PNA2t/DNAt1 triplexes (two PNAt probes with one DNA) were prepared by mixing the PNAt probe (60 µL, 200 µM) and DNAtc or DNAt1 sequence (60 µL, 100 µM) in Milli-Q water (78 µL) with the addition of NaCl (2 µL, 500 mM). The mixture was heated to 90 °C for 5 min in an incubator and slowly cooled down to room temperature. The duplex and triplex solutions of 30 µM each were formed and stored at -20 °C for further use. Optimization of TO Intercalation. A volume of 50 µL of the as-prepared PNAd/DNAdc or PNA2t/DNAtc solution was mixed (29) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188–1196.

Scheme 2. Strategy of Polymer Amplified TO Emission As the Signal for Sequence Specific DNA Detection Using Label Free PNA, S1 Nuclease, and PFP

with 50 µL 0.5× S1 reaction buffer and further diluted to 300 µL with Milli-Q water. The pH was then adjusted to 7.4 with aqueous sodium hydroxide (0.6 µL, 2 M). TO (1 × 10-4 M) was added dropwise to each complex solution at intervals of 2 µL. After each addition, the mixture was gently shaken at room temperature before fluorescence measurement. The excitation wavelength was 488 nm, and the fluorescence was collected in the range of 500-700 nm. The optimized amount of TO required for maximum intercalation was studied based on the TO fluorescence intensity change at 530 nm. Enzyme Digestion. For all digestion reactions, 50 µL of 0.5× S1 reaction buffer was mixed with 50 µL of as-prepared PNAd/ DNAdc, PNAd/DNAd1, PNA2t/DNAtc, or PNA2t/DNAt1 solution. After addition of S1 nuclease (0.69 µL, 95 u/µL), the digestion of PNAd/DNAdc and PNAd/DNAd1 duplexes (5 µM) was conducted at 24 °C for different times, while the digestion of PNA2t/DNAtc and PNA2t/DNAt1 triplexes was conducted at 37 °C for different times. The optimized amount of TO (2.7 µM for duplexes or 3.3 µM for triplexes) was added at one shot, and the TO emission intensity at 530 nm was monitored upon direct excitation of TO at 488 nm. The digestion time was optimized to give the lowest signal for mismatches and maintain a high signal for fully complementary sequences. FRET Experiments. TO (2.7 µM) was added to 5 µM PNAd/ DNAdc after digestion at 24 °C for 20 min, or 3.3 µM TO was added to 5 µM PNA2t/DNAtc after digestion at 37 °C for 5 min, which was followed by the addition of PFP dropwise at intervals of 0.9 µM. The mixture was gently shaken for 2-3 s after each addition. The solution spectra were collected in the range of 380-700 nm upon excitation of PFP at 365 nm. The spectrum for the same amount of free PFP was used as the reference. The amount of PFP was optimized to achieve the maximal sensitized TO emission intensity. SNP Detection. Each of PNAd/DNAdc or PNAd/DNAd1 duplexes as well as PNA2t/DNAtc or PNA2t/DNAt1 triplexes (50 µL, 30 µM) were mixed with 50 µL of 0.5× S1 reaction buffer. After addition of S1 nuclease (0.69 µL, 95 u/µL), the digestion was conducted at 24 °C for the duplexes (20 min) and 37 °C

for the triplexes (5 min). The solution was then diluted to 300 µL with Milli-Q water, and the pH was adjusted to 7.4 with aqueous sodium hydroxide (0.6 µL, 2 M). The optimized amount of TO was then added to each solution. The TO emission intensity at 530 nm was monitored upon direct excitation at 488 nm. This was followed by the addition of the optimized amount of PFP to each solution. The PFP sensitized TO emission was collected upon excitation at 365 nm, and the photos were taken by setting the cuvettes under a hand-held UV-lamp. RESULTS AND DISCUSSION The principle of TO based sequence specific detection is based on the selective enzyme digestion of DNA sequences that are not perfectly matched to the PNA probe, which is followed by TO intercalation and signal amplification. It has been reported that oligonucleotides with lengths and sequences that could perfectly match the PNA probes are efficiently protected from nuclease S1 digestion.6b On the other hand, the presence of mismatches could lead to DNA conformational changes and the resultant physicochemical perturbation at the PNA/DNA mismatch site should facilitate the access of S1 nuclease for DNA digestion. This hypothesis was confirmed by Komiyama et al., who demonstrated that a one-base alternation in DNA sequence could be detected in terms of the difference in digestion efficiency.8,19 Since S1 nuclease has been widely used for ssDNA digestion, standard DNA digestion protocols have been directly adapted to selectively digest PNA/DNA complexes that contain mismatches. The signal amplification is also performed according to the protocols developed in our group.27b The general detection scheme (Scheme 2) contains a label free PNA probe (shown in red), S1 nuclease (shown in orange), an intercalating dye (TO, shown in green), and a cationic polyfluorene (shown in yellow). In general, PNA and DNA can form either duplexes or triplexes, dependent on the probe sequences.25,30 To (30) Seog, K. K.; Peter, E. N.; Michael, E.; Ole, B.; Rolf, H. B.; Bengt, N. J. Am. Chem. Soc. 1993, 115, 6477–6481.

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Figure 1. TO emission intensities for PNAd/DNAdc duplex (blank) and PNA2t/DNAtc triplex (shadow) solutions after dropwise addition of TO at intervals of 2 µL. [PNAd/DNAdc or PNA2t/DNAtc] ) 5.0 × 10-6 M. TO stock concentration is 1 × 10-4 M, λexc ) 488 nm. The emission intensities were collected at 530 nm.

demonstrate the generality of the detection method, in this work, two different sequences of PNA probes were selected. One was a homopyrimide probe, PNAt, which was reported to form PNAt2/ DNAtc triplexes, involving two PNAt probes and one DNAtc molecule per complex.25,30 The other probe (PNAd) contains both purine and pyrimide bases, which could only form PNAd/ DNAdc duplexes. One starts with a PNA probe in solution. Upon addition of the target, hybridization between PNA and DNA occurs. If the target DNA is perfectly complementary to the PNA probe in solution, the PNA probe protects the DNA from hydrolysis by S1 nuclease, leaving the negatively charged PNA/ DNA complexes in solution. On the other hand, the mismatched DNA is not sufficiently protected by the PNA probe and is hydrolyzed into small fragments by the enzyme. In the next step, when TO is added to the solution, the TO fluorescence is only detectable in the presence of PNA/DNA complexes due to intercalation. When PFP is further added, the polymer could electrostatically associate with the PNA/DNA complexes. Excitation of PFP leads to energy transfer from the polymer to TO, resulting in sensitized TO emission, and the solution fluorescence appears yellow. In the case of mismatched sequences, almost no PNA/DNA duplexes remain in solution after enzyme digestion and TO intercalation is not possible. The distance between PFP and TO remains too large for efficient energy transfer, and the solution color remains blue. Optimization of TO Intercalation. The optimization of TO intercalation was first studied in PNAd/DNAdc duplex solution before digestion. The annealed PNAd/DNAdc (50 µL) was mixed with 50 µL 0.5× S1 buffer (pH ) 4.6), and the pH was then adjusted to 7.4 with aqueous sodium hydroxide (0.6 µL, 2 M). The as prepared buffer is used for all the following experiments for TO intercalation after S1 digestion. Different amounts of TO were then added to the solution, and the TO emission intensity at 530 nm was monitored. As shown in Figure 1, the TO emission intensity in the duplex solution (blank bar) increases with the increased TO concentration up to 2.7 × 10-6 M. Further addition of TO to the duplexes leads to a slight decrease in fluorescence intensity, which indicates the saturation of TO intercalating at [TO] ) 2.7 × 10-6 M. Optimization 4102

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of TO intercalation for the same concentration of PNAt2/DNAtc triplexes was also studied, and the maximum TO emission intensity was observed at [TO] ) 3.3 × 10-6 M (shadowed bar in Figure 1). The emission spectra for both duplex and triplex solutions with the highest TO emission intensity are shown in the Supporting Information as Figure S1. The optimized TO amount was used in the following experiments for a digestion time study and FRET experiments for DNA detection. Enzyme Digestion. To optimize the conditions for discriminating the perfectly matched complexes from mismatched ones, several samples of 5 µM PNAd/DNAdc or PNAd/DNAd1 were prepared and the digestion was conducted at 24 °C for different periods of time. This was followed by the addition of 2.7 µM TO to each solution, and the TO emission intensity at 530 nm was monitored upon excitation at 488 nm. Figure 2a shows the time-dependent changes in the ratio of TO emission intensity obtained after digestion (I) to that before digestion (Io). For PNAd/DNAdc complexes, there is less than a 5% decrease in TO emission intensity after digestion for 20 min. However, the decrease in TO emission intensity is over 60% and 95% after digestion of PNAd/DNAd1 for 10 min and 20 min, respectively. Under the same digestion conditions, changes in TO emission signal for PNAt2/DNAt1 solutions are less obvious as compared to that for PNAd/DNAd1 (Figure S2 in the Supporting Information). After increasing the temperature to 37 °C, very weak TO emission is left in solution for PNA2t/DNAt1 triplexes after digestion for 5 min (shown in Figure 2b). In addition, comparison of the TO emission intensity for PNA2t/DNAtc before and after digestion at 37 °C for 5 min reveals that there is less than a 10% decrease in TO emission, which indicates that the DNAtc is effectively protected by the PNAt. Figure 3 shows the photoluminescence (PL) spectra for solutions of PNAd/DNAdc and PNAd/DNAd1 (5.0 µM each) after digestion at room temperature for 20 min, which were followed by addition of 2.7 µM TO and excitation at 488 nm. The TO emission in PNAd/DNAdc duplex solution is much more intense than that obtained in PNAd/DNAd1 solution. The low TO emission from PNAd/DNAd1 duplex solution is due to nonspecific interaction between TO and small digested fragments. A similar trend is also observed for the triplex system (Figure S3 in the Supporting Information). In addition, the changes in TO emission intensity for DNAt/DNAtc solutions before and after digestion were also studied. Before digestion, for solutions containing 2.7 × 10-6 M TO and 5.0 µM DNAt/DNAtc or PNAd/ DNAdc, the fluorescence intensity of TO for the DNA duplex is over 10-fold more intense as compared to that from the PNAd/DNAdc solution, due to the high binding affinity between TO and DNAt/DNAtc duplexes (Figure S4 in the Supporting Information). However, there is only very weak fluorescence from TO in the DNAt/DNAtc complex solution after S1 nuclease digestion (green curve in Figure 3). This result indicates that the DNAt/DNAtc complexes are effectively hydrolyzed by S1 nuclease under the operating conditions. This agrees with the literature report that low concentrations of S1 nuclease can digest single-stranded DNAs or RNAs, while double-stranded

Figure 2. The time-dependent changes in the ratio of TO emission intensity obtained after digestion (I) to that before digestion (Io) for (a) PNAd/DNAdc vs PNAd/DNAd1 and (b) PNAt2/DNAtc vs PNAt2/DNAt1 solutions. Digestion was conducted at 24 °C for duplexes and 37 °C for triplexes. [PNAd/DNAdc (blank), PNAd/DNAd1 (shadow), PNA2t/DNAtc (blank), PNA2t/DNAt1 (shadow)] ) 5.0 × 10-6 M, 2.7 × 10-6 M TO for duplexes and 3.3 × 10-6 M TO for triplexes, λexc ) 488 nm. The emission intensities were collected at 530 nm.

Figure 3. PL spectra of TO in duplex solutions after digestion. [PNAd/ DNAdc (black), PNAd/DNAd1 (red), DNAt/DNAtc (green)] ) 5.0 × 10-6 M, [TO] ) 2.7 × 10-6 M, λexc ) 488 nm.

nucleic acids (DNA/DNA, DNA/RNA, and RNA/RNA) could be degraded by large concentrations of enzyme.31 These experimental data indicate that the presence of a complementary DNA sequence in solution could be detected by simply measuring the TO fluorescence in solution after digestion. This is because both PNA/DNA complexes containing mismatches and DNA duplexes are digested except for the perfectly matched PNA/DNA complexes. Although there is a difference in TO emission in the presence of complementary and one-base mismatched DNA sequence, the detection sensitivity for both duplexes and triplexes is strictly constrained by the low fluorescence intensity of intercalated TO. The sensitivity of this detection system could be improved through introducing an energy donor to improve the intercalated TO emission. FRET Experiments and Visual Detection. Figure 4 shows the absorption and photoluminescence (PL) spectra for both PFP and TO in aqueous solution. PFP shows an emission maximum at 421 nm, and TO has an absorption maximum at 501 nm. There is an obvious spectral overlap between the emission of PFP and (31) http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/nucleases.html

Figure 4. Absorption and PL spectra of PFP solution (O) and TO (b) in freshly prepared TO/DNA aqueous solution.

the absorption spectrum of TO, which should favor FRET from the donor (PFP) to the acceptor (TO).32 With the optimized amount of TO in 5 µM PNAd/DNAdc duplex or PNA2t/DNAtc triplex solutions, PFP was added dropwise to serve as the energy donor for sensitized TO emission. The corresponding spectra are shown in Figure 5a. The PFP concentration dependent changes of TO emission intensity at 540 nm after polymer emission tail subtraction are shown in Figure 5b. For PNAd/DNAdc duplexes, upon excitation of the polymer at 365 nm, the TO emission intensity increases with increasing [PFP] in solution, and the maximum TO emission signal is observed at [PFP] ) 23 µM. Further addition of PFP results in a slight decrease in TO emission intensity mainly due to the electrostatic repulsion between cationic PFP and TO molecules which leads to reduced TO intercalation.27b Similarly, 20-23 µM PFP was added to the PNAt2/DNAtc triplex solution to yield the highest polymer sensitized TO emission (Figure S5 in the Supporting Information). In both cases, it is noteworthy that the TO emission intensity upon excitation of PFP at 365 nm is approximately 19-fold greater than that upon direct excitation of TO at 488 nm in the absence of polymer, indicating the signal amplification provided by PFP. The greatly elevated (32) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/ Plenum Publisher: New York, 1999.

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Figure 5. (a) Emission spectra of solutions containing PNAd/DNAdc duplexes after digestion and TO intercalation, which was followed by the addition of PFP at intervals of 3.3 × 10-6 M. Direct excitation of TO at 488 nm is also shown for comparison (dark red). (b) The PFP concentration dependent changes of TO emission intensity at 540 nm after polymer emission tail subtraction. [PNAd/DNAdc] ) 5.0 × 10-6 M, [TO] ) 2.7 × 10-6 M, λexc ) 365 nm.

Figure 6. (a) The normalized PL spectra for PNAd/DNAdc, (black) PNAd/DNAd1 (red), and DNA/DNAtc (green) duplex solutions after digestion and addition of the optimized amount of TO and PFP. The PL spectrum for PFP solution is shown in blue. The solution fluorescent color for PNAd/DNAdc (1), PNAd/DNAd1 (2), and DNA/DNAtc (3) is shown in the inset of part a. (b) TO emission intensity at 540 nm after polymer emission tail subtraction. [PNAd/DNAdc (1), PNAd/DNAd1 (2), and DNA/DNAtc (3)] ) 5.0 × 10-6 M, [TO] ) 2.7 × 10-6 M, [PFP] ) 2.3 × 10-5 M, λexc ) 365 nm.

Figure 7. The PFP sensitized TO emission intensities in solutions containing varied amount of DNAdc (a) and DNAtc (b). The data are based on the average of three independent experiments. [TO]/[PFP]/[duplex] ) 2.7:23:5 or [TO]/[PFP]/[triplex] ) 3.3:23:5.

signal in the presence of PFP can dramatically increase the detection sensitivity and reduce the detection errors compared with the assay utilizing TO alone as the fluorescence reporter. 4104

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Figure 6a shows the PL spectra for PNAd/DNAdc, PNAd/ DNAd1, and DNA/DNAtc solutions after digestion with nuclease S1 for 20 min and with the addition of the optimized concentra-

tions of TO and PFP. Figure 6b illustrates the obtained TO emission intensity at 540 nm after subtracting the PFP emission tail. Contrary to the high intensity of TO for PNAd/DNAdc duplex solutions, the signal for PNAd/DNAd1 and DNA/DNAtc solutions remains low. This result indicates that the small fragments formed after digestion cannot bind to TO efficiently or form complexes with PFP. Comparison of the data between Figure 6b and Figure 3 reveals that the detection sensitivity is significantly improved in the presence of PFP as compared to that with TO only as the signal reporter. The difference in solution fluorescence for PNAd/DNAdc and PNAd/DNAd1 in the presence of TO and PFP is readily distinguishable by a naked-eye under a hand-held UV lamp with an excitation wavelength of 365 nm. The inset in Figure 6a shows the photos for solutions containing an initial PNAd/DNAdc, PNAd/DNAd1, and DNA/DNAtc concentration of 5 µM, after digestion and addition of the optimized amount of TO and PFP. For PNAd/DNAdc duplexes, the solution fluorescence appears yellow due to energy transfer from PFP to TO. On the other hand, DNA sequences in PNAd/DNAd1 and DNA/DNAtc are hydrolyzed by S1 nuclease and the solutions remaining have blue fluorescence. A similar result is also obtained for the triplex system (Figure S6 in the Supporting Information). As a result, a readily visual detection is achieved to discriminate the perfect complementary DNA sequence in solution, with a detection limit of 5 µM. Limit of Detection for DNA by a Fluorometer. To further investigate the detection sensitivity of the developed DNA sensing strategy by a fluorometer, different amount of DNAdc and DNAtc sequences were annealed with PNAd and PNAt probes, and S1 digestion was conducted at 24 °C for duplexes (20 min) and 37 °C for triplexes (5 min). The proportional amount of TO and PFP was then added to each solution at a ratio of [TO]/ [PFP]/[duplex] ) 2.7:23:5 or [TO]/[PFP]/[triplex] ) 3.3:23:5, according to the optimal conditions obtained in the previous sections. Figure 7 shows the standard calibration curve for PFP sensitized TO emission intensity in solution as a function of the complementary DNA concentration. The detection limit is estimated to be ∼0.25 µM for DNAdc and DNAtc, respectively. This calibration curve could be used for DNA quantification in real samples since all the nonspecific complexes could be digested, and the TO emission are due to the intercalation of TO into perfectly matched PNA/DNA complexes.

CONCLUSION In summary, we developed a simple and highly selective label free DNA sequence-specific detection system, which is generally applicable to DNA detection with PNA probes. The detection system takes advantage of the distinctly decreased stability of PNA/DNA complexes caused by base mutation in DNA sequence and the fact that mismatched DNA could be efficiently digested by S1 nuclease to small fragments. The perfectly complementary DNA sequence is sufficiently protected by the PNA probe, which ensures the intercalation of TO and turns on the solution fluorescence. Furthermore, the negatively charged PNA/DNA complexes are able to form complexes with cationic lightharvesting conjugated polymers through electrostatic interactions, and energy transfer from CCP to the intercalated TO further enhances the detection signal. Compared with other fluorescence based DNA detection methods, this assay forsakes the complex design and high cost in modifying probes with signaling dyes. In addition, the PNA based enzyme digestion strategy also represents one of the very few examples that an intercalation dye could be used for sequence specific DNA detection. The assay has shown a detection limit of 0.25 µM using a standard fluorometer. In addition, this sensing strategy allows visual detection of target DNA with a detection limit of 5 µM, which provides a fast and reliable detection of specific DNA sequences with good sensitivity and SNP selectivity. ACKNOWLEDGMENT The authors are grateful to the National University of Singapore (Grants ARF R-279-000-197-112/133, R-279-000-233-123, and R-279000-234-123), Ministry of Education (Grant R-279-000-255-112), and Temasek Defence Systems Institute (Grants R-279-000-268-422, R-279-000-268-592, R279-000-268-232) for financial support. K. Li thanks NUS for support via a research fellowship. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 23, 2009. Accepted April 1, 2009. AC9003985

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