Electrostatic Contributions to Cyanine Dye Aggregation on Peptide

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Langmuir 2003, 19, 6449-6455

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Electrostatic Contributions to Cyanine Dye Aggregation on Peptide Nucleic Acid Templates† Miaomiao Wang, Isil Dilek, and Bruce A. Armitage* Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890 Received February 7, 2003. In Final Form: April 9, 2003 A symmetrical benzothiazole-based dicarbocyanine dye with anionic propylsulfonate substituents was synthesized in order to assess the importance of electrostatic forces in the assembly of helical dye aggregates on peptide nucleic acid (PNA) templates (Smith, J. O.; Olson, D. A.; Armitage, B. A. J. Am. Chem. Soc. 1999, 121, 2686-2695). UV-vis and circular dichroism spectroscopy demonstrate effective aggregation of the dye on a 10 base pair PNA-PNA duplex but weak aggregation on the analogous PNA-DNA duplex. The CD spectra are consistent with a left-handed helical morphology for the dye aggregate, as expected for the left-handed PNA-PNA double helical template. Continuous variations analysis indicates formation of a 6:1 aggregate of the dye on a 10 base pair PNA-PNA duplex. An analogous dye bearing two cationic side chains and a net charge of +3 does not aggregate on the PNA-PNA duplex but can be recruited into a mixed aggregate with the anionic dye due to partial charge neutralization. Finally, charged groups at the termini of the duplex affect both the stability and chirality of the dye aggregate, with cationic L-lysine residues promoting formation of stable, left-handed helical aggregates, while anionic L-glutamate residues lead to less stable, right-handed helical aggregates.

Introduction

* To whom correspondence should be addressed. Tel: (412) 2684196. Fax: (412) 268-1061. E-mail: [email protected]. † This article is dedicated to the memory of David O’Brien, who exemplified integrity, professionalism, and creativity.

While many of the gross structural properties of duplex DNA are maintained when a PNA-DNA hybrid is formed, an interesting distinction arises from the failure of most traditional DNA-binding small molecules to bind to PNAcontaining hybrids.17 The interactions between small molecules and DNA have been intensively investigated in recent years, due to their utility in DNA detection, as modulators of gene expression, and as potential therapeutic agents. Many of these molecular recognition events are noncovalent and are characterized by varying degrees of binding affinity and sequence specificity.18,19 The noncovalent forces that contribute to binding include hydrogen bonding, the hydrophobic effect, van der Waals attractions, and electrostatic interactions, since the ligands are most often cationic. If one or both of the strands are replaced by PNA, electrostatic interactions between the small molecules and the negative DNA backbone are reduced or lost. Wittung et al. studied various DNA intercalators and minor groove binders with DNA-DNA, PNA-DNA, and PNA-PNA.17 Their results show that the minor groove binders, distamycin A and DAPI (diamidinophenylindole), bind only weakly to certain PNA-DNA duplexes possessing the AATA motif. No binding to PNA-PNA duplexes was observed. Meanwhile, intercalators exhibited no binding to PNA-DNA or PNA-

(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1498-1500. (2) Nielsen, P. E. Pure Appl. Chem. 1998, 70, 105-110. (3) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998, 37, 2796-2823. (4) Corey, D. R. Trends Biotechnol. 1997, 15, 224-229. (5) Nielsen, P. E.; Haaima, G. Chem. Soc. Rev. 1997, 26, 73-78. (6) Egholm, M.; Buchardt, O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature 1993, 365, 566-568. (7) Wittung, P.; Nielsen, P. E.; Buchardt, O.; Egholm, M.; Norde´n, B. Nature 1994, 368, 561-563. (8) Tomac, S.; Sarkar, M.; Ratilainen, T.; Wittung, P.; Nielsen, P. E.; Norde´n, B.; Gra¨slund, A. J. Am. Chem. Soc. 1996, 118, 5544-5552. (9) Ratilainen, T.; Holme´n, A.; Tuite, E.; Haaima, G.; Christensen, L.; Nielsen, P. E.; Norde´n, B. Biochemistry 1998, 37, 12331-12342. (10) Ratilainen, T.; Holme´n, A.; Tuite, E.; Nielsen, P. E.; Norde´n, B. Biochemistry 2000, 39, 7781-7791. (11) Erikkson, M.; Nielsen, P. E. Nat. Struct. Biol. 1996, 3, 410-413. (12) Rasmussen, H.; Kastrup, J. S.; Nielsen, J. N.; Nielsen, J. M.; Nielsen, P. E. Nat. Struct. Biol. 1997, 4, 98-101.

(13) Hanvey, J. C.; Peffer, N. J.; Bisi, J. E.; Thomson, S. A.; Cadilla, R.; Josey, J. A.; Ricca, D. J.; Hassman, C. F.; Bonham, M. A.; Au, K. G.; Carter, S. G.; Bruckenstein, D. A.; Boyd, A. L.; Noble, S. A.; Babiss, L. E. Science 1992, 258, 1481-1485. (14) Good, L.; Nielsen, P. E. Antisense Nucleic Acid Drug Dev. 1997, 7, 431-437. (15) Good, L.; Awasthi, S. K.; Dryselius, R.; Larsson, O.; Nielsen, P. E. Nat. Biotechnol. 2001, 19, 360-364. (16) Nielsen, P. E.; Egholm, M. Peptide Nucleic Acids. Protocols and Applications; Nielsen, P. E., Egholm, M., Eds.; Horizon Scientific Press: Norfolk, 1999. (17) Wittung, P.; Kim, S. K.; Buchardt, O.; Nielsen, P. E.; Norde´n, B. Nucleic Acids Res. 1994, 22, 5371-5377. (18) Wilson, W. D. Reversible Interactions of Nucleic Acids with Small Molecules. In Nucleic Acids in Chemistry and Biology; Blackburn, G. M., Gait, M. J., Eds.; Oxford University Press: Oxford, 1996; pp 329374. (19) Mountzouris, J. A.; Hurley, L. H. Small Molecule-DNA Interactions. In Bioorganic Chemistry: Nucleic Acids; Hecht, S. M., Ed.; Oxford University Press: New York, 1996; pp 288-323.

Peptide nucleic acid (PNA)1 is a synthetic mimic of DNA, in which the sugar-phosphodiester backbone of DNA is replaced by poly-N-(2-aminoethyl)glycine.2-5 The natural nucleobases are attached to the glycine nitrogen via carbonyl methylene linkers. PNA hybridizes with complementary DNA, RNA, or PNA strands according to the Watson-Crick rules.6,7 The resulting double- or triplehelical complexes exhibit greater thermal and thermodynamic stabilities than analogous DNA-DNA duplexes due in part to the lack of electrostatic repulsion between the PNA strand and the complementary strand.6,8-10 Highresolution structural studies show that PNA-DNA11 and PNA-PNA12 duplexes are wider and have larger helical pitches (i.e., are underwound) relative to canonical B-form DNA/DNA duplexes. The interest in PNA ranges from applications in molecular biology, antisense gene therapy, and antibiotics to fundamental biomolecular recognition mechanisms.13-16

10.1021/la0342161 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/22/2003

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Wang et al.

electrostatic complementarity to drive supramolecular assembly. In addition to modifying the dye structure, we synthesized PNA oligomers bearing anionic glutamate residues on each terminus. Thus, the two PNA-PNA duplexes shown in Chart 1 will have either two positive charges on each end for a net charge of +4 or two negative charges on each end for a net charge of -4. These terminal charges are shown to affect not only the stability of the templated dye aggregate but also the absolute chirality of the structure. Experimental Section

PNA duplexes. The failure of intercalators to bind to PNAcontaining hybrids remains unexplained. In contrast to these results, we recently demonstrated the high-affinity binding of the cyanine dye DiSC2(5) (Chart 1) to PNA-PNA and PNA-DNA duplexes and a PNA-DNA-PNA triple helix.20 The dye did not bind to these structures as an isolated monomer but rather spontaneously assembled into a helical aggregate using the PNA as a template. This binding mode results in an instantaneous color change from blue to purple, providing a simple indicator for PNA hybridization,21,22 and two genetic screening methods based on this phenomenon were recently reported.23,24 The generality of this method is underscored by the observation that DiSC2(5) aggregates on all PNA-DNA sequences tested, including random mixtures of purines and pyrimidines. This stands in contrast to our experiments with DNA templates, where the dye aggregates only on alternating A-T and I-C sequences.25,26 In contrast to the monocationic DiSC2(5), the tricationic analogue DiSC3+(5) (Chart 1) exhibited no affinity for either PNA-PNA or PNA-DNA.20 We reasoned that this was due to insurmountable electrostatic repulsions that would be present in an aggregate of DiSC3+(5), especially on PNA-PNA, where no negative charges are present to compensate for the positively charged dye. High concentrations of DiSC3+(5) successfully aggregate in the minor groove of certain DNA-DNA sequences, where the negative charge density is quite high due to the phosphate groups present at every position on both strands.26,27 To provide greater insight into the role of electrostatics in cyanine dye recognition of PNA-containing hybrids, we synthesized the anionic analogue DiSC3-(5) (Chart 1). This dye maintains the same chromophore as the other two dyes but features two anionic propylsulfonate substituents. Thus, while the substituents of individual dyes within an aggregate should repel one another, the net charge on each dye, -1, has the same magnitude as that on DiSC2(5). This dye readily assembles into chiral aggregates on PNA-PNA templates either alone or in combination with the tricationic dye, forming mixed aggregates. The latter case is an example of using (20) Smith, J. O.; Olson, D. A.; Armitage, B. A. J. Am. Chem. Soc. 1999, 121, 2686-2695. (21) Kushon, S. A.; Jordan, J. P.; Seifert, J. L.; Nielsen, P. E.; Nielsen, H.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 10805-10813. (22) Datta, B.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 96129619. (23) Wilhelmsson, L. M.; Norde´n, B.; Mukherjee, K.; Dulay, M. T.; Zare, R. N. Nucleic Acids Res. 2002, 30, e3. (24) Komiyama, M.; Ye, S.; Liang, X.; Yamamoto, Y.; Tomita, T.; Zhou, J.-M.; Aburatani, H. J. Am. Chem. Soc. 2003, 125, 3758-3762. (25) Seifert, J. L.; Connor, R. E.; Kushon, S. A.; Wang, M.; Armitage, B. A. J. Am. Chem. Soc. 1999, 121, 2987-2995. (26) Wang, M.; Silva, G. L.; Armitage, B. A. J. Am. Chem. Soc. 2000, 122, 9977-9986. (27) Cao, R.; Venezia, C. F.; Armitage, B. A. J. Biomol. Struct. Dyn. 2001, 18, 844-856.

Materials. t-Boc-protected PNA monomers were purchased from Applied Biosystems, Inc. PNA oligomers were synthesized according to standard solid-phase synthesis protocols.28,29 They were purified by high-performance liquid chromatography (HPLC), and the masses were determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) (P1, H-GTA-GAT-CAC-T-Lys-NH2 calculated mass 2856.8, observed mass 2859.5; P2, H-AGT-GAT-CTA-C-Lys-NH2 calculated mass 2856.8, observed mass 2855.9; P3, Ac-Glu-GTA-GAT-CAC-TGlu-NH2 calculated mass 3027.9, observed mass 3032.3; P4, AcGlu-AGT-GAT-CTA-C-Glu-NH2 calculated mass 3027.9, observed mass 3030.9). No internal standard was used in the mass spectrometer. DNA (purified by gel filtration chromatography) was purchased from Integrated DNA Technologies and used as received. PNA and DNA stock solutions were prepared in deionized water, and the concentrations were determined spectrophotometrically using extinction coefficients calculated from DNA nearest-neighbor values or PNA monomer values. DiSC3+(5) and DiSC2(5) were purchased from Molecular Probes, Inc., and were used without further purification. (DiSC2(5) is no longer available from this vendor but can be purchased from Aldrich Chemical Co.) Stock solutions of dyes were prepared in methanol and filtered through glass wool. The concentrations were determined in methanol using either the manufacturers’ extinction coefficients (651 ) 260 000 M-1 cm-1 for DiSC2(5), 653 ) 160 000 M-1 cm-1 for DiSC3+(5)) or the experimentally determined value (650 ) 191 605 M-1 cm-1 for DiSC3-(5)). DiSC3-(5) was synthesized following similar conditions reported by Southwick and co-workers:30 0.350 g (1.0 mmol) of 2-methyl-1-(3-propylsulfonate)-benzothiazolium inner salt, kindly provided by Dr. Gloria Silva, was dissolved in 10 mL of methanol. 1,3,3-Trimethoxypropene (0.134 g, 1.0 mmol) was added to the solution, followed by potassium acetate (0.200 g, 2.0 mmol). The system was refluxed in a water bath for 1 h; then heating was removed and the resulting mixture was allowed to dry overnight at room temperature. The dark purple powder was triturated with methanol and purified by column chromatography using a chloroform/methanol eluent. The solvent was removed by rotary evaporation and, after overnight drying, yielded 0.256 g (0.369 mmol, 36.9% yield) of DiSC3-(5). Electrospray MS (observed mass, 577.13; calculated mass, 577.75 for M- - K+) and elemental analysis (calculated for C25H25N2O6S4K‚5H2O: C, 42.48; H, 4.99; N, 3.96; S, 18.14. found: C, 43.39; H, 5.20; N, 3.97; S, 17.87) are consistent with the expected product. 1H NMR (300 MHz, DMSOd6): 7.61-8.01 (m, 6H, 2-Ar-H(4H) and β-CHd (2H)), 7.307.61 (m, 4H, 1-Ar-H), 6.77 (d, 2H, R-CHd), 6.54 (t, 1H, γ-CHd), 4.62 (t, 4H, 1′-CH2-), 2.74 (t, 4H, 3′-CH2-), 2.16 (m, 4H, 2′CH2-). Equipment. UV-vis measurements were performed on a Varian Cary3 spectrometer equipped with a thermoelectrically controlled multicell holder. Circular dichroism (CD) measurements were recorded on a JASCO J-715 spectropolarimeter equipped with a thermoelectrically controlled single cell holder. (28) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen, H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; Coull, J.; Berg, R. H. J. Pept. Sci. 1995, 3, 175-183. (29) Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz, H. G.; Otteson, K.; Ørum, H. J. Pept. Res. 1997, 49, 80-88. (30) Southwick, P. L.; Ernst, L. A.; Tauriello, E. W.; Parker, S. R.; Mujumdar, R. B.; Mujumdar, S. R.; Clever, H. A.; Waggoner, A. S. Cytometry 1990, 11, 418.

Cyanine Dye Aggregation on Templates

Figure 1. Normalized UV-vis spectra of DiSC3-(5) (dotted line), DiSC3+(5) (dashed line), and DiSC2(5) (solid line) in water. [Dye] ) 6.0 µM. Dye Spectroscopic Experiments. PNA-PNA (PP) and PNA-DNA (PD) duplexes were prepared by mixing together equimolar amounts of the complementary strands in a buffer containing 10 mM sodium phosphate (pH 7.5), heating to 75 °C, and then cooling slowly (1 °C/min) to room temperature prior to spectroscopic experiments. Dye stock solutions were added in 1.0 µM aliquots up to 10 µM. Spectra were recorded after equilibrating the sample for 5 min at 20 °C. For experiments involving mixtures of DiSC3-(5) and DiSC3+(5), samples were first prepared containing DiSC3-(5) and PP. After recording UV-vis or CD spectra, an equimolar amount of DiSC3+(5) was added to the sample, which was then heated to 60 °C and cooled back to 20 °C prior to recording the spectrum. This annealing step was necessary to achieve formation of the mixed aggregate. Temperature-Dependent Spectroscopy. In temperaturedependent UV-vis spectra, samples containing 6.0 µM dye and 1.0 µM duplex in 10 mM sodium phosphate buffer (pH 7.5) were cooled to 15 °C prior to acquiring spectra. Subsequent spectra were recorded at 5 °C intervals up to 60 °C. For melting curves, samples containing 4.0 µM total dye concentration and 1.0 µM duplex in 10 mM sodium phosphate buffer and either 10% or 20% methanol were first cooled to 15 °C and then heated to 60 °C at 1 °C/min. The samples were then cooled back to 15 °C at the same rate. Absorbance values at 540 nm were recorded at 0.5 °C intervals. Methanol was used in these samples in order to suppress the melting temperatures. For PNA-PNA duplex melting curves, samples containing 1.0 µM of each strand in 10 mM sodium phosphate (pH 7.5) were heated to 95 °C for 5 min and then cooled to 15 °C followed by reheating to 95 °C at a rate of 1 °C/min. Absorbance values at 260 nm were recorded at 0.5 °C intervals. Melting temperatures were determined from maxima observed in first-derivative plots. Continuous Variations Experiment. Samples containing various ratios of DiSC3-(5) and PP with a total concentration of 5.0 µM were prepared in 10 mM sodium phosphate buffer (pH 7.5). UV-vis spectra were recorded at 20 °C, and the absorbance at 540 nm was plotted versus the mole fraction of the dye.

Results The three cyanine dyes shown in Chart 1 have identical chromophores, leading to similar absorption spectra in methanol, featuring maxima at 650 nm and vibrational shoulders at 580 nm. However, the structural differences begin to manifest themselves when the spectra are recorded in pure water (Figure 1). A blue-shifted band is observed for DiSC2(5) and DiSC3-(5), but not DiSC3+(5). This band is assigned to a face-to-face dimer structure based on literature reports.25,31 The shoulders evident in the spectrum for the tricationic dye DiSC3+(5) are more likely due to vibrational bands for the dye rather than to

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aggregation since similar features are observed in methanol, where the cyanines do not dimerize. DiSC2(5) and DiSC3-(5) have net charges of +1 and -1, respectively, while DiSC3+(5) bears a charge of +3. The weaker dimerization of the latter dye is most likely due to enhanced intermolecular Coulombic repulsions. Thus, while dimerization of cyanine dyes in water is driven by the hydrophobicity and polarizability of the dye,31,32 electrostatics can be used to tune the strength of the intermolecular interaction through the N-substituents. This phenomenon is explored below for the aggregation of cyanine dyes on PNA templates. Binding of DiSC3-(5) to PNA/PNA and PNA/DNA Duplexes. The PP and PD duplexes used for these experiments (Chart 1) are the same as those used in our previous study involving DiSC2(5).20 Note that the PNA sequences have cationic N-termini due to ammonium groups. The C-termini of the PNAs are also cationic, by virtue of an L-lysine residue incorporated as the first unit during the solid-phase synthesis procedure. Since the PNAs were synthesized on a methylbenzhydrylamine (MBHA) resin, the final product is a C-terminal amide, rather than a carboxylate. As shown in Figure 2A, titration of DiSC3-(5) into PNA-PNA duplex PP results in only one significant UVvis absorption band at 540 nm, which we assign to a PNAbound aggregate of DiSC3-(5) based on the similarity of this feature to our previous findings with DiSC2(5).20 The 650 nm monomer peak is very weak and only grows bigger when the dye concentration exceeds 6 µM (i.e., a ratio of 6 dyes/duplex). Titration of DiSC3-(5) into the PNADNA duplex PD having the same sequence as PP results in the spectra shown in Figure 2B, which exhibit two major peaks. The monomer peak at 650 nm is much more intense than observed with PP, while the peak observed at 580 nm is assigned to a dimeric form of DiSC3-(5). Since this dye also dimerizes in the absence of PNA-DNA, we cannot determine from these results whether the dimer is bound to the duplex or free in solution. However, CD spectra indicate that the dimer is in fact not bound to PD (vide infra). The aggregate peak observed at 540 nm in PP only appears as a weak shoulder in the spectra for PD, indicating that aggregation of DiSC3-(5) is considerably less favorable on the PNA-DNA duplex. To determine the binding stoichiometry of DiSC3-(5) to PP, a continuous variations experiment was performed, in which the dye plus duplex total concentration was kept constant but the individual concentrations were varied.33 Figure 3 shows the resulting Job plot based on the aggregate absorbance (A540). As shown, the absorbance increased as the fraction of DiSC3-(5) increased, until Xdye reached 0.85, which corresponds to approximately 6 dye molecules per duplex, consistent with the titration data in Figure 2A. The plot does not simply fall to zero after passing Xdye ) 0.85 but appears to exhibit a second inflection at approximately Xdye ) 0.90, corresponding to a 9:1 complex. This observation suggests that more than 6 dyes can bind to a given duplex but that these higher order aggregates are of lower affinity since the 6:1 complex is clearly observed in the Job plot. We obtained similar results for the monocationic DiSC2(5) on the PNA-DNA duplex PD.20 By calculating the remaining unbound DiSC3-(5) in PP from the UV-vis absorbance at 650 nm and assuming a 6:1 stoichiometry, we estimate the equilibrium binding constant for DiSC3-(5) on PP to be ca. 1040 M-6. (31) West, W.; Pearce, S. J. Phys. Chem. 1965, 69, 1894-1903. (32) Herz, A. H. Photogr. Sci. Eng. 1974, 18, 323-335. (33) Job, P. Ann. Chim. (Paris) 1928, 9, 113-203.

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Figure 2. UV-vis titration of DiSC3-(5) into PP (A) or PD (B) duplexes in 10 mM sodium phosphate buffer (pH ) 7.5). Duplex concentrations were 1.0 µM, and DiSC3-(5) was added in 1.0 µM aliquots.

Figure 3. Job plot for determining DiSC3-(5)-PP binding stoichiometry. The total concentration of DiSC3-(5) and PP was kept constant at 5.0 µM. The absorbance at 540 nm (A540) is plotted versus the mole fraction of dye. Data points represent mean and standard deviation for three separate measurements.

Binding of DiSC3-(5) to PP can also be seen in Figure 4, which shows the CD spectrum of DiSC3-(5) in the presence of the PNA-PNA duplex. Cyanine dyes bound to PNA-containing hybrids assemble into helical aggregates where the chirality of the “template” hybrid is transferred to the dye aggregate.20 The chirality of PNAPNA duplexes is complicated since helical induction is completely controlled by the C-terminal L-lysine residues.7,34,35 Subtle changes in sequence can produce substantial changes in the CD spectrum of PNA-PNA duplexes.34 Nevertheless, duplex PP has been assigned as a left-handed helix,35 consistent with our previous (34) Wittung, P.; Erikkson, M.; Lyng, R.; Nielsen, P. E.; Norde´n, B. J. Am. Chem. Soc. 1995, 117, 10167-10173. (35) Lagriffoule, P.; Wittung, P.; Erikkson, M.; Jensen, K. K.; Norden, B.; Buchardt, O.; Nielsen, P. E. Chem.sEur. J. 1997, 3, 912-919.

Figure 4. CD spectra of 6.0 µM DiSC3-(5) bound to 1.0 µM PP (solid line) or PD (dashed line). The spectra represent the averages of eight scans recorded at a rate of 100 nm/min.

findings for DiSC2(5) and our current work with DiSC3-(5), where a strong left-handed helical exciton splitting is observed on the PNA-PNA duplex. The exciton splitting is due to the interactions of two or more neighboring chromophores,36 which also demonstrates the aggregation of multiple dye molecules on the PP template rather than binding as isolated monomers. A more complex spectrum is observed for DiSC3-(5) in the presence of the PNA-DNA duplex (Figure 4). The splitting pattern does not immediately suggest a structural model for how the dye binds to PD, although the lack of a well-defined absorption at 580 nm indicates that the dimer band observed in the UV-vis spectra (Figure 2B) is due to unbound dye. The generally poor binding of this dye to PNA-DNA led us to focus our efforts on characterizing the aggregates formed on the PNA-PNA template. (36) Nakanishi, K.; Berova, N.; Woody, R. W. Circular Dichroism: Principles and Applications; VCH: New York, 1994.

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Figure 5. Temperature-dependent UV-vis spectra of 6.0 µM DiSC3-(5) with 1.0 µM PP. Spectra were recorded at 5 °C intervals from 15 to 60 °C. Inset: Temperature-dependent absorbance curve recorded at 540 nm for 6.0 µM DiSC3-(5) with 1.0 µM PP. The buffer contained 10 mM NaPi (pH ) 7.5) and 10% methanol.

Figure 6. UV-vis spectra recorded for 1.0 µM (A) and 6.0 µM (B) DiSC3-(5) in the presence of either PP (solid) or PPGlu (dashed) duplexes. [Duplex] ) 1.0 µM.

Aggregation of DiSC2(5) on PNA templates is a highly cooperative process and is characterized by a strong temperature dependence.20 This is also true for DiSC3-(5) (Figure 5), where the aggregate is observed even at the highest temperatures. Spectra were not recorded above 60 °C due to thermal degradation of the dye. A thermal denaturation (“melting”) curve is shown in the inset to Figure 5. Note that 10% methanol was included as a cosolvent because this destabilizes the aggregate, allowing the transition to occur at temperatures where degradation of the dye is negligible. A cooperative transition is observed, and differentiating the melting curve yields the

transition temperature Tm ) 50.4 °C for the aggregate. Note that the Tm for the dye aggregate is lower than the temperature at which the PP duplex denatures to single strands (66 °C), demonstrating that the thermal stability of the dye aggregate is independent of the thermal stability of the PNA template. Recording the melting curves using both heating and cooling ramps demonstrates a significant hysteresis in the assembly and disassembly of the dye aggregate. Specifically, the Tm determined from an experiment where the A540 was monitored as the sample cooled was 38.3 °C, that is, 12.1 °C lower than the value determined when the

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Figure 7. CD spectra recorded for 1.0 µM (circles) and 6.0 µM (triangles) DiSC3-(5) in the presence of either PP (filled symbols) or PPGlu (open symbols) duplexes. [Duplex] ) 1.0 µM.

Figure 8. CD spectra recorded for 1.0 µM PP (solid line) and PPGlu (dashed line) duplexes.

sample was heated (data not shown). The lower Tm observed when cooling the sample likely illustrates the slow kinetics involved in assembling the 6:1 aggregate of the dye on the duplex. Effect of Template Charge on Dye Aggregation. The results shown in Figures 2 and 4 illustrate that an anionic PNA-DNA template inhibits aggregation by DiSC3-(5). To further investigate the role of template charge in controlling aggregation, we synthesized complementary PNA strands with anionic glutamate residues at each end and with the N-termini capped by reaction with acetic anhydride. Thus, the duplex PPGlu bears a net charge of -4 due to the presence of a negative charge on both ends of both strands (Chart 1). UV melting curves recorded for PP and PPGlu demonstrate that the tetracationic and tetraanionic duplexes have comparable thermal and thermodynamic stabilities (Figure S1 in the Supporting Information). UV-vis spectra for two concentrations of DiSC3-(5) in the presence of either PP or PPGlu are shown in Figure 6. At the low concentration, much greater aggregation is observed for the cationic duplex PP based on the stronger

Wang et al.

Figure 9. UV-vis spectra showing titration of DiSC3-(5) into a solution containing 6.0 µM DiSC3+(5) and 1.0 µM PP duplex. DiSC3-(5) was added in 1.0 µm aliquots, and spectra were recorded at 20 °C.

band at 540 nm. However, at the higher concentration, the two duplexes give similar levels of aggregation, indicating that the cooperativity inherent to aggregation can overcome Coulombic repulsion by the terminal glutamate residues. This stands in contrast to the experiments comparing PP with PD (Figure 2), where weak aggregation by the dye on the PNA-DNA duplex was observed over the entire concentration range. CD spectra recorded under the same conditions are shown in Figure 7. At both concentrations, the spectra are more intense for the cationic duplex. What is also interesting about these spectra is their near mirror-image relationship depending on the duplex. This indicates that the helical aggregates have opposite chirality, that is, lefthanded on PP and right-handed on PPGlu. Since the dye is achiral, this distinction most likely arises from the PNA-PNA duplexes. To confirm this, CD spectra were recorded in the UV region without any dye present (Figure 8). While the intensities are different, presumably due to the presence of four chiral L-glutamate residues on PPGlu versus only two L-lysine groups on PP, the spectra appear as near mirror images, indicating that the two duplexes indeed have opposite chirality. Wittung et al. have reported similar findings for PNA-PNA duplexes terminated with glutamate or lysine residues.34 Electrostatic Complementarity Promotes Mixed Aggregate Formation on PNA-PNA. The N-substituents on DiSC3-(5) and DiSC3+(5) lead to drastically different PNA-binding behavior. While the anionic dye aggregates readily on PP, interaction of the tricationic dye with the PNA-PNA duplex is extremely weak, based on the very minor perturbation evident in the UV-vis spectrum (Figure S2 in the Supporting Information). Although DiSC3-(5) clearly has much higher affinity for the duplex, we reasoned that the anionic dye might assist binding of the tricationic dye through charge neutralization within a mixed aggregate. Since the two dyes have identical chromophores, this would simply require that DiSC3-(5) interact more strongly with DiSC3+(5) than with itself, a reasonable expectation given the electrostatic complementarity between the substituents on the two dyes. Figure 9 illustrates the effect of titrating DiSC3-(5) into a solution containing DiSC3+(5) and PP. If the anionic dye were to aggregate on the duplex without incorporating the tricationic dye into the assembly, one would expect to

Cyanine Dye Aggregation on Templates

Figure 10. Comparison of CD spectra of DiSC3-(5) (3.0 µM, dashed line) or DiSC3-(5) and DiSC3+(5) (3.0 µM each, solid line) in 1.0 µM PP.

see the monomer band at 650 nm increase slightly as the DiSC3-(5) concentration increases (Figure 2A). Instead, this band decreases as the anionic dye is titrated into the sample. This can only be explained by incorporation of DiSC3+(5) into a mixed aggregate with DiSC3-(5), although the results do not establish whether the mixed aggregate is assembled on the PNA duplex or in solution. This issue is resolved by comparing the CD spectra of 3 µM DiSC3-(5) in the absence and presence of 3 µM DiSC3+(5) (Figure 10). Addition of the tricationic dye increases the intensity of the spectrum and also alters the ratio of the positive and negative peaks from 1.62 to 1.92. Note that DiSC3+(5) alone gives no CD signal in this region (data not shown). These spectral changes indicate that the mixed aggregate is assembled on the PNA-PNA duplex template rather than in solution. Discussion Aggregation of cyanine dyes on PNA-containing hybrid duplexes and triplexes yields an instantaneous visible color change, regardless of base sequence, which is useful for qualitative analysis of PNA hybridization to complementary DNA.20-22 While efforts to develop this discovery into a DNA detection method are underway,23,24 it is unclear if sufficient sensitivity will be achieved to compete with existing technologies that detect femtomolar concentrations of DNA with high sequence specificity.37 The experiments reported here are of a more fundamental nature and were designed to probe the role of Coulombic forces in assembling the helical dye aggregate. This was done through variation of the charge either on the dye or on the PNA hybrids. The anionic cyanine dye DiSC3-(5) spontaneously assembles into stable left-handed helical aggregates on the cationic PNA-PNA duplex PP. The temperature and concentration dependencies of this process, along with the chirality of the resulting helical aggregate, are similar to what we previously reported for the cationic dye DiSC2(5). However, incorporation of negative charges into the duplex, either uniformly (PD) or solely at the termini (PPGlu), significantly destabilizes the aggregate. For the PNA-DNA duplex, where negative charges are present at each position, only weak aggregation occurs, even at the highest dye concentration. The fact that more aggregation is observed on longer duplex templates suggests (37) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1539.

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that the aggregate assembles along the full length of the duplex. Thus, repulsions at each base pair step should have a greater impact on the assembly of an aggregate from a negatively charged dye. In the case of PPGlu, higher dye concentrations effectively overcome Coulombic repulsions from the terminal glutamate side chains, leading to stable aggregates. The last approach to addressing Coulombic forces in the assembly of PNA-templated aggregates involved the use of electrostatic complementarity, which has been used in other contexts to promote selective assembly of noncovalent structures involving small molecules,38 peptides,39 proteins,40 and synthetic polymers.41 We find that combining DiSC3-(5) and DiSC3+(5) leads to formation of a PNA-templated mixed aggregate of the two dyes, although the tricationic dye exhibits virtually no aggregation in the absence of the anionic dye. Finally, the observation of opposite chiralities for the aggregates templated on PP and PPGlu is intriguing. The CD spectra recorded in the absence of dye indicate that the two duplexes twist in opposite directions, as proposed previously for PP and an analogue of PPGlu lacking the N-terminal glutamate residues.34 No structural model has been put forth to explain why terminal L-lysine and L-glutamate residues would induce opposite helicities for PNA-PNA duplexes. A number of PNA oligomers have been synthesized with chiral modifications in the backbone.35,42,43 It would be interesting to analyze the impact of these modifications on the helicity of the resulting duplexes and dye aggregates. The ability of cyanine dyes to adopt helical morphologies upon either spontaneous or templated aggregation constitutes a supramolecular assembly phenomenon having both fundamental and applied facets. In addition to the colorimetric assay for DNA detection cited above, chiral chromophore aggregates should have interesting nonlinear optical properties. Variation of the nucleic acid template, cyanine dye structure, and environmental conditions can lead to a diverse range of supramolecular morphologies. Our current efforts are devoted to understanding the dye-template interactions at the atomic level. Acknowledgment. This material is based upon work supported by the National Science Foundation under Grant Number CHE-0078765. We are grateful to Dr. Gloria Silva for providing a key starting material for the synthesis of DiSC3-(5), to Bhaskar Datta, Lorraine Hsu, and Terry Watt for PNA synthesis, and to Eleanor Muise for technical assistance. Mass spectra were measured in the Center for Molecular Analysis at Carnegie Mellon University, supported by NSF CHE-9808188. Supporting Information Available: (1) UV melting curve of PP and PPGlu duplexes and (2) UV-vis spectra comparison of DiSC3-(5) and DiSC3+(5) in the presence of PP. This material is available free of charge via the Internet at http://pubs.acs.org. LA0342161 (38) Haack, T.; Peczuh, M. W.; Salvatella, X.; Sanchez-Quesada, J.; de Mendoza, J.; Hamilton, A. D.; Giralt, E. J. Am. Chem. Soc. 1999, 121, 11813-11820. (39) Kennan, A. J.; Haridas, V.; Severin, K.; Lee, D. H.; Ghadiri, M. R. J. Am. Chem. Soc. 2001, 123, 1797-1803. (40) Hendsch, Z. S.; Nohaile, M. J.; Sauer, R. T.; Tidor, B. J. Am. Chem. Soc. 2001, 123, 1264-1265. (41) Zhai, L.; McCullough, R. D. Adv. Mater. 2002, 14, 901-905. (42) Haaima, G.; Lohse, A.; Buchardt, O.; Nielsen, P. E. Angew. Chem., Int. Ed. Engl. 1996, 35, 1939-1942. (43) Sforza, S.; Corradini, R.; Ghirardi, S.; Dossena, A.; Marchelli, R. Eur. J. Org. Chem. 2000, 2905-2913.