J. Phys. Chem. 1996, 100, 9181-9186
9181
Binding of TO-PRO-3 and TOTO-3 to DNA: Fluorescence and Hole-Burning Studies N. Milanovich, M. Suh, R. Jankowiak, G. J. Small, and J. M. Hayes* Department of Chemistry and Ames Laboratory, Iowa State UniVersity, Ames, Iowa 50011 ReceiVed: January 2, 1996; In Final Form: March 19, 1996X
The thiazole orange derivative, TO-PRO-3, and its dimeric analogue, TOTO-3, bind strongly to both doubleand single-stranded DNA. Both the bound and the free dye undergo efficient nonphotochemical hole burning. From the structure of the hole burned and the fluorescence line-narrowed spectra, it is concluded that there are multiple modes of binding of the dyes to DNA. In one mode, the absorption, fluorescence, and holeburned vibronic band structure are similar to those of the free dye. It is proposed that the molecules in this mode are externally bound to the DNA chain. Molecules in the other binding mode are characterized by a strong interaction with DNA. These are proposed to be molecules in intercalated or base-stacked configurations.
Introduction The dyes TO-PRO-3 and TOTO-3 (structures shown in Figure 1) are the reddest absorbing and emitting members of a family of DNA staining dyes based on thiazole orange and oxazole yellow. The dyes in this family consist of a benzothiazole or benzoxazole ring linked to a quinoline ring through a conjugated chain. The length of the chain is indicated by the numerical suffix in the trivial names of the dyes; e.g., TO-PRO-3 has a three-carbon conjugated chain. Increasing the length of the chain shifts the dye’s absorption and emission to the red. The dimeric forms of these dyes, i.e., TOTO and YOYO, use a biscationic linker to join two monomers at the quinoline rings. The absorption and fluorescence spectra of the dyes are little changed by dimerization, but the binding constants to DNA increase greatly, e.g., by a factor of 40 in the case of TO-PRO-3 and TOTO-3.1 This increased binding strength is thought to be due to the fact that the dyes primarily bind to DNA by intercalation and the dimeric forms are bis-intercalators. The binding is further stabilized by electrostatic interaction of the bis-cationic linker with the minor groove of the DNA.2 The high binding constants, coupled with a large increase in fluorescence quantum yield for the bound dyes relative to that of the free dye, have led to numerous applications of these dyes to the sensitive detection of DNA. With the monomeric dyes, Zhu et al.3 demonstrated sensitivities of 2-4 amol of DNA base pairs per band in capillary electrophoresis (CE) separations of DNA fragments. For the monomeric dyes, on-column staining was used with the dye added to the running buffer. The excellent sensitivity obtained is due to the low quantum efficiency of fluorescence from the unbound dye. In the same paper, CE separation of fragments prestained with the dimeric dyes was also reported. Broadening of the CE bands due to differences in the number of dye molecules per fragment was eliminated by the addition of another intercalating agent (9aminoacridine) to the running buffer. Although sensitivities were not as good as for the monomeric dyes, they were still quite good. Rye et al.4 have also demonstrated picogram detection limits of DNA fragments prestained with dimeric dyes with lengths ranging from 600 to 24 000 base pairs (bp) in gel electrophoresis separations using a confocal gel scanner for detection. The monomeric and dimeric dyes have also been used in flow cytometry5,6 and in confocal laser microscopy of paraffin sections.7 X
Abstract published in AdVance ACS Abstracts, May 1, 1996.
S0022-3654(96)00062-7 CCC: $12.00
Figure 1. (bottom).
Molecular structures of TO-PRO-3 (top) and TOTO-3
Despite the proliferation of applications of these dyes, little attention has been devoted to an understanding of their basic spectroscopy. NMR spectroscopy has been used to determine preferred base sequences for binding of TOTO-1 to oligomers8 and for a structure determination of the same dye to one oligomer.9 In those studies, it was found that the sequence CTAG bound the dye with only one conformation. The structure determination was for an octamer with CTAG as its central sequence. In that oligomer, TOTO-1 was bis-intercalated with a local distortion of the DNA structure in the intercalation region.9 For oligomers with other sequences, more than one binding conformation was detected, although structures for these cases were not determined.8 Optical spectroscopy has been applied most extensively to the YO and YOYO dyes by Carlsson and co-workers.10-13 They have used linear and circular dichroism and absorption and emission spectroscopy to study both the free dyes and the dyes © 1996 American Chemical Society
9182 J. Phys. Chem., Vol. 100, No. 21, 1996
Milanovich et al.
bound to ds-DNA. They also have used these techniques to investigate band-broadening and band-splitting in gel electrophoresis separations of fragments stained with the bis-intercalators YOYO-1, TOTO-1, and ethidium dimer12 and sequence preferences in synthetic polynucleotides.13 In this paper, we report results of low-temperature spectroscopic measurements (absorption and fluorescence) on TOPRO-3 and TOTO-3 both free and bound to double-stranded and single-stranded DNA. Low-temperature is used so that the high-resolution techniques of hole burning and fluorescence linenarrowing can be applied to these systems. These techniques have been shown to be extremely powerful in the elucidation of the details of molecular interactions in a variety of biological materials.14 Hole-burning of dyes bound to DNA oligonucleotides has previously been reported by Flo¨ser and Haarer15 and by Chang et al.16,17 Experimental Section TO-PRO-3 and TOTO-3 iodide were obtained from Molecular Probes, Inc., as 1 mM solutions in DMSO and were used without further purification. Double-stranded (ds) and singlestranded (ss) calf thymus DNA were from Sigma Chemical Co. Phosphate buffer was used for the DNA sample preparations (20 mM disodium phosphate, 100 mM NaCl, pH 7.0, filtered through a 0.22 µm pore size filter for sterilization). DNA concentrations were determined by measuring the absorbance at 258 nm. The following extinction coefficients were used:18 ds calf thymus DNA, 6.6 × 103 M-1 cm-1; ss calf thymus DNA, 1.0 × 104 M-1 cm-1. TO-PRO-3 samples for absorption measurements were prepared by adding the dye to a 30% water/ 70% glycerol mixture to give a dye concentration of 30 µM. TO-PRO-3/DNA samples were made by adding dye to buffered DNA solution and then diluting with glycerol to give dye and DNA concentrations of 30 and 600 µM, respectively, base pairs in 40% glycerol/60% buffer. For fluorescence excitation and emission measurements, dye and DNA concentrations were reduced to 3 and 60 µM, respectively. In both cases the dye to DNA base pair ratio was 0.05. For TOTO-3 absorption measurements, 50 µM solutions in a 1:4:5 (v/v/v) DMSO/water/ glycerol mixture were used. For bound TOTO-3 samples 10 µM dye was mixed with 500 µM DNA in 60% buffer/40% glycerol (dye to base pair ratio, 0.02). For TOTO-3 fluorescence measurements, the dye concentration was reduced to 5 µM and the DNA concentration increased to 900 µM. Absorption spectra were measured with a Bruker IFS-120 HR Fourier transform spectrometer over the range 10 00025 000 cm-1. The spectra were measured with 1 cm-1 resolution for the hole-burning measurements and with 4 cm-1 resolution for simple absorption measurements. The samples were in either a 1 mm path length cell formed from two quartz plates separated by a 1 mm spacer or in 2 mm i.d. quartz tubes. Samples were generally cooled slowly to 77 K by cold nitrogen vapor and then immersed into liquid helium. The sample temperature was measured with a silicon diode mounted on the cell holder. Hole-burning was done with the output from a Coherent 699-29 ring dye laser using DCM Special dye (Exciton Chemical Co.) pumped by a 6 W argon ion laser. The laser line width was 1, the electronphonon interaction is designated as being strong. The values
Figure 7. FLN spectra of TO-PRO-3 bound to single-stranded calf thymus DNA at dye/DNA base pair ratios of 0.05 (A), 0.1 (B), and 0.2 (C). Note the broad band peaked at ∼15 150 cm-1.
2.4 and 2.5 in Table 2 are consistent with intercalation in which the electronic transition incorporates a large degree of charge transfer character due to the proximity and geometry of the DNA bases. The values of 0.85 and 0.53, on the other hand, are similar to those of the free dye and indicate a molecular geometry in which the chromophore is accessible to solvent, i.e., an externally bound complex. In Figure 7 are shown line-narrowed fluorescence spectra for TO-PRO-3 bound to ss-DNA at various binding ratios. Note that at all binding ratios there is a broad fluorescence component that peaks at ∼660 nm. This feature is coincident with the peak of the 77 K fluorescence shown in Figure 2B. The narrow lines in the spectrum arise from fluorescence from the externally bound dye molecules. As the d/bp ratio increases, the intensity of the narrow features increases relative to the intensity of the broad peak. This would indicate that for TO-PRO-3, with ssDNA, the “base-stacked” sites are filled in preference to the externally bound sites. This is in contrast to the YO-PRO results which indicated that the two binding constants are similar.11 We note that spectra of the type shown in Figure 7 are not without precedent. Low-temperature, high-resolution (FLN) spectroscopy has been used to identify all of the binding modes of enantiomeric forms of diol epoxides of benzo[a]pyrene.23 Benzo[a]pyrene diol epoxide (BPDE) is a metabolite of the potent carcinogen benzo[a]pyrene. It exists as two diastereomeric forms, each of which can be resolved into (+) and (-) enantiomers. The mutagenic and carcinogenic properties of the various forms vary greatly. The carcinogenic properties arise from covalent bonding of the diol epoxide to the exocyclic amine groups of adenine and guanine bases in DNA. From low-temperature spectroscopy of DNA adducted with BPDE, Lu et al. identified two different conformations of the BPDE chromophore.23 These were identified as an externally bound adduct and an interior (intercalated) adduct. Subsequently, solution NMR and molecular modeling were used to determine the structures of the four enantiomers with oligonucleotides.24,25 FLN spectra of these confirmed the original interpretation of Lu et al. that the sharp features observed in the spectra arose from externally bound forms while the red-shifted, broad features were due to intercalated adducts.27 The FLN spectra shown in Figure 7 are quite similar to those of the BPDE/DNA adducts; i.e., one can clearly observe zero-phonon lines in the high-energy region (from externally complexed chromophore) superimposed on a broad fluorescence band originating from the strongly base-stacked complexes. Although there is a great deal of similarity between the spectra of TO-PRO-3 and TOTO-3 bound to DNA and BPDE adducts with DNA, it is important to point out that for the BPDE adducts there is a
9186 J. Phys. Chem., Vol. 100, No. 21, 1996 covalent bond between the BPDE and the DNA, while for the TO-PRO-3 (TOTO-3) case the dye forms a physical complex with the DNA. In the latter case, there is no covalent bond formed. The interactions between dye and DNA are primarily electrostatic or involve hydrogen bonding. In conclusion, the hole and line-narrowed fluorescence spectra clearly show contributions from two binding modes of TOPRO-3 and TOTO-3 with DNA. In contrast to the reported binding of YOYO and YO-PRO,11 for TO-PRO-3, the externally bound conformer is present at all concentrations. On the basis of the near coincidence of the absorption maxima of the bound and free dyes, the externally bound conformation could even be the preferred mode of binding, with the shift of fluorescence maxima being due to an increased fluorescence quantum yield for this conformation. Acknowledgment. We acknowledge the assistance of Sergei Savikhan and To¨nu Reinot in measuring the fluorescence lifetimes. The Ames Laboratory is operated for the U.S. Department of Energy by Iowa State University under Contract W-7405-Eng-82, and this work was supported by the Office of Health and Environmental Research, Office of Energy Research. References and Notes (1) Haugland, R. P. Molecular Probes: Handbook of Fluorescence Probes and Research Chemicals; Molecular Probes, Inc.: Eugene, OR, 1992; p 221. (2) Rye, H. S.; Yue, S.; Wemmer, D. E.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1992, 20, 2803. (3) Zhu, H.; Clark, S. M.; Benson, S. C.; Rye, H. S.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1994, 66, 1941. (4) Rye, H. S.; Yue, S.; Quesada, M. A.; Haugland, R. P.; Mathies, R. A.; Glazer, A. N. Methods Enzymol. 1993, 217, 414. (5) Hirons, G. T.; Fawcett, J. J.; Crissman, H. A. Cytometry 1994, 15, 129. (6) Van Hooijdonk, C. A. E. M.; Glade, C. P.; Van Erp, P. E. J. Cytometry 1995, 7, 185. (7) Tekola, P.; Baak, T. P. A.; Belie¨n, J. A. M.; Brugghe, J. Cytometry 1994, 17, 191.
Milanovich et al. (8) Jacobsen, J. P.; Pedersen, J. B.; Hansen, L. F.; Wemmer, D. E. Nucleic Acids Res. 1995, 23, 753. (9) Spielmann, H. P.; Wemmer, D. E.; Jacobsen, J. P. Biochemistry 1995, 34, 8542. (10) Carlsson, C.; Larsson, A.; Jonsson, M.; Albinsson, B.; Norde´n, B. J. Phys. Chem. 1994, 98, 10313. (11) Larsson, A.; Carlsson, C.; Jonsson, M.; Albinsson, B. J. Am. Chem Soc. 1994, 116, 8459. (12) Carlsson, C.; Jonsson, M.; Åkerman, B. Nucleic Acids Res. 1995, 23, 2413. (13) Larsson, A.; Carlsson, C.; Jonsson, M. Biopolymers 1995, 36, 153. (14) For reviews see: Johnson, S. G.; Lee, I.-J.; Small, G. J. In Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, FL, 1993; p 199. Reddy, N. R. S.; Lyle, P. A.; Small, G. J. Photosyn. Res. 1992, 31, 169. Jankowiak, R.; Small, G. J. In The Photosynthetic Reaction Center; Diesenhofer, J., Norris, J., Eds.; Academic: New York, 1993; Vol. II, p 133. Jankowiak, R.; Small, G. J. Chem. Res. Toxicol. 1991, 4, 256. (15) Flo¨ser, G.; Haarer, D. Chem. Phys. Lett. 1988, 147, 288. (16) Chang, T.-C.; Chiang, C.-C.; Chou, S.-H.; Peck, K. J. Phys. Chem. 1995, 99, 6620. (17) Chang, T.-C.; Chiang, C.-C.; Peck, K. J. Am. Chem. Soc. 1995, 117, 7576. (18) Wells, R. D.; Larson, J. E.; Grant, R. C.; Shortle, B. E.; Cantor, C. R. J. Mol. Biol. 1970, 54, 465. (19) Shu, L.; Small, G. J. Chem. Phys. 1990, 141, 447. (20) Shu, L.; Small, G. J. J. Opt. Soc. Am. 1992, 9, 724. (21) Kenney, M. J.; Jankowiak, R.; Small, G. J. Chem. Phys. 1990, 146, 47. (22) Milanovich, N.; Suh, M.; Hayes, J. M.; Small, G. J. Biospectroscopy 1996, in press. (23) Jankowiak, R.; Lu, P.; Small, G. J.; Geacintov, N. E. Chem. Res. Toxicol. 1990, 3, 39. (24) Cosman, M.; de los Santos, C.; Fiala, R.; Hingerty, B. E.; Singh, S. B.; Ibanez, V.; Margulis, L. A.; Live, D.; Geacintov, N. E.; Broyde, S.; Patel, D. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 1914. (25) de los Santos, C.; Cosman, M.; Hingerty, B. E.; Ibanez, V.; Margulis, L. A.; Geacintov, N. E.; Broyde, S.; Patel, D. J. Biochemistry 1992, 31, 5245. (26) Cosman, M.; de los Santos, C.; Fiala, R.; Hingerty, B. E.; Ibanez, V.; Luna, E.; Harvey, R. G.; Geacintov, N. E.; Broyde, S.; Patel, D. J. Biochemistry 1993, 32, 4145. (27) Suh, M.; Ariese, F.; Small, G. J.; Jankowiak, R.; Liu, T.-M.; Geacintov, N. E. Biophys. Chem. 1995, 56, 281.
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