Stacking of meso-Tetrakis(3-N-methylpyridiniumyl)porphyrin on Poly[d

Biao Jin , Jung Eun Ahn , Jae Hong Ko , Wei Wang , Sung Wook Han and Seog K. Kim ... Jong Heon Shin , Yoon Mo Hwang , Yoon Jung Jang , Seog K. Kim...
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17106

J. Phys. Chem. B 2004, 108, 17106-17111

Stacking of meso-Tetrakis(3-N-methylpyridiniumyl)porphyrin on Poly[d(A-T)2]: Importance of the Distance between Porphyrin’s Positive Charges Taegi Park,† Jong Sub Shin,† Sung Wook Han,‡ Jong-Keun Son,§ and Seog K. Kim*,† Department of Chemistry and College of Pharmacy, Yeungnam UniVersity, Dae-dong, Kyoungsan City, Kyoung-buk 712-749, and Department of EnVironmental Engineering, Kyoungwoon UniVersity, Sangdong-myun, Kumi, Kyoung-buk 136-701, Republic of Korea ReceiVed: June 21, 2004; In Final Form: August 17, 2004

The meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (m-TMPyP)-poly[d(A-T)2] complex at a [porphyrin]/ [DNA base] ratio of 0.05 was investigated by polarized spectroscopy, including circular and linear dichroism (CD and LD). The spectroscopic properties of the complex depend on the NaCl concentration and time, and can be classified into three types. At a NaCl concentration below 10 mM, a strong bisignate CD spectrum and negative LD spectrum in the Soret band were apparent immediately after mixing. As time lapsed, the spectral properties changed and the complex exhibited a small bisignate CD spectrum and a strong negative LD spectrum. On the other hand, at a NaCl concentration above 10 mM, the bisignate feature of the CD spectrum in the porphyrin absorption region exhibited little change. The LD signal in the DNA absorption region collapsed at this condition, while the CD signal was retained, indicating that DNA remained in the B-form but was compactly aggregated. Surprisingly, a strong positive band in the Soret region was apparent, and its magnitude increased with time. The observed spectral properties for the m-TMPyP-poly[d(A-T)2] complex are in contrast with those of the meso-tetrakis(4-N-methylpyridiniumyl)porphyrin-poly[d(A-T)2] complex, which binds at the groove of a polynucleotide at a low NaCl concentration and stacks at a high NaCl concentration. The difference in the binding mode may be attributed to the difference in distance between negatively charged phosphate groups: this distance is variable in the m-TMPyP molecule.

Introduction Binding of water-soluble cationic porphyrins to DNA has been a subject of intensive study for its potential application to biology and photodynamic therapy. Various factors, including the nature of DNA, the central metal, and peripheral substituents of the porphyrin ring, have been known to affect the binding modes of the porphyrin-DNA complex.1-3 While a representative of the porphyrin family, meso-tetrakis(4-N-methylpyridiniumyl)porphyrin (referred to as p-TMPyP in this work) intercalates between the base pairs of GC-rich DNAs,4-7 it either stacks along the DNA stem or binds at the groove of AT-rich DNA.7-13 The groove binding mode, which is represented by a positive circular dichroism in the same absorption region, prefers a low [porphyrin]/[DNA] ratio and a high salt concentration. Self-assembly or aggregation of porphyrin on the DNA template, which is represented by a conservative bisignate CD signal in the Soret absorption band, was noticed almost from the outset.14,15 These porphyrin species were assigned to a moderate stacking mode (or modest aggregation). On the other hand, it has also been found that certain porphyrin derivatives can form extended assemblies (or extensive stacking) on the nonaggregated DNA templates.16-18 The stacked or assembled porphyrin is characterized by a bisignate CD signal in the Soret band. Stacking of p-TMPyP has recently been shown to occur in the major groove.19,20 * To whom correspondence should be addressed. Tel: + 82 53 810 2362. Fax: + 82 53 815 5412. E mail: [email protected]. † Department of Chemistry, Yeungnam University. ‡ Kyoungwoon University. § College of Pharmacy, Yeungnam University.

Figure 1. Molecular structures of meso-tetrakis(N-methylpyridinium3-yl)porphyrin and meso-tetrakis(N-methylpyridinium-4-yl)porphyrin, respectively, referred to as m-TMPyP and p-TMPyP in the text.

The nature of the peripheral “tentacle” substituents and the electron richness of the porphyrin core also affect the stacking vs intercalation vs groove binding mode as well as the extent of aggregation.4,21,22 On the other hand, we recently have reported that a small change in the position of the N-methyl group at the peripheral pyridinium ring, i.e., in comparison between p-TMPyP and meso-tetrakis(3-N-methylpyridiniumyl)porphyrin (referred to as m-TMPyP in this work, Figure 1), resulted in a contrasting binding mode. The m-TMPyP exhibited an extensive stacking mode upon binding to poly[d(A-T)2], while p-TMPyP binds at the groove of the same polynucleotide.23,24 To understand the abnormal behavior of m-TMPyP, the effect of the salt concentration on the stacking of m-TMPyP as well as the groove binding of p-TMPyP to poly[d(A-T)2] was thoroughly studied in this work using polarized spectros-

10.1021/jp0473117 CCC: $27.50 © 2004 American Chemical Society Published on Web 10/07/2004

Stacking of m-TMPyP on Poly[d(A-T)2]

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copy including circular and linear dichroism (referred to as CD and LD, respectively). Materials and Methods Both p- and m-TMPyP were purchased from Midcentury (Chicago, IL) and used without further purification. The extinction coefficients were measured as 424nm ) 2.26 × 105 cm-1 M-1 and 417nm ) 2.78 × 105 cm-1 M-1 for p- and m-TMPyP, respectively, in 5 mM cacodylate buffer, pH 7.0, which was used throughout this work. Therefore, 0 mM NaCl in this work indicates that 5 mM Na+ is present in the solution that comes from the counterion of the cacodylate molecule. Consequently, 100 mM NaCl indicates 105 mM Na+ ion. Polynucleotides were purchased from Amersham Biosciences (Piscataway, NJ) and dissolved in 5 mM cacodylate buffer containing 100 mM NaCl and 1 mM EDTA, pH 7.0, followed by several rounds of dialysis against 1 mM cacodylate buffer solution, pH 7.0, at 4 °C. Their concentrations were determined using the extinction coefficients 254nm ) 8400 cm-1 M-1 and 262nm ) 6600 cm-1 M-1 for poly[d(G-C)]2 and poly[d(A-T)2, respectively. The appearance of the spectra for the porphyrinDNA complex may be affected by the order of mixing.25 Therefore, aliquots of concentrated porphyrin were always added last to the DNA solution. Absorption spectra were recorded on a Jasco V550 (Tokyo, Japan). CD spectroscopy is a particularly useful tool for investigating porphyrin-DNA interactions. A negative CD band in the Soret absorption region generally has been accepted as a diagnostic for the intercalated porphyrin, while porphyrin that bound at the groove exhibits a positive CD band in the same region. A bisignate CD band is pronounced for the stacked porphyrins. Although the origin of this CD signal is not fully understood, interactions between the electric transition moment of porphyrin and those of chirally arranged DNA bases cause the induced CD for the monomeric porphyrin-DNA complex. On the other hand, electronic coupling between porphyrins that stacked along the DNA stem has been proposed.17 CD spectra were measured on a Jasco J715 (Tokyo, Japan) spectropolarimeter using a 1 cm cell. The CD spectrum was averaged over an appropriate number of scans when necessary. LD is the difference in anisotropic absorption of light polarized in planes parallel and perpendicular to the direction of orientation. Measured LD is divided by isotropic absorption to give reduced LD (LDr), which is related to the angle, R, between the transition moment of drugs and the macroscopic orientation axis, and the orientation factor, S, such that S ) 1 for a sample perfectly oriented parallel to the flow direction and S ) 0 for an isotropic orientation.26,27 LD on the floworiented porphyrin-DNA complexes was measured using a Jasco J715 spectropolarimeter equipped with an inner rotating Couette cell. The path length for the LD measurement was 1 mm. Results In this work, the ratio [porphyrin]/[DNA base] was fixed at 0.05. At this mixing ratio, in the p-TMPyP-polynucleotide complex cases, the binding mode is believed to be homogeneous. The spectral properties of the m-TMPyP-poly[d(A-T)2] complex changed with time until 3 h after mixing. Although timebased spectral changes are interesting, we limited ourselves to investigating the binding mode of the m-TMPyP-poly[d(AT)2] complex immediately after mixing and after the complex is stabilized (after 4 h).

Figure 2. Absorption (a), LD (b), and CD (c) spectra of the m-TMPyP-poly[d(A-T)2] complex immediately (solid curve, curve a) and 4 h (dotted curve, curve b) after mixing in the presence of 0 mM NaCl. In panels a and b, the absorption spectrum of DNA-free m-TMPyP and the LD spectrum of porphyrin-free poly[d(A-T)2] are shown (dashed curve, curve c), respectively, for comparison. The concentration of poly[d(A-T)2] is 100 µM in the base, and that of m-porphyrin is 5 µM.

Spectral Properties of the m-TMPyP-Poly[d(A-T)2] Complex at Low and High NaCl Concentrations. Although the spectral properties of the m-TMPyP-poly[d(A-T)2] complex at a low NaCl concentration have been reported already,23,24 it will be necessary to repeat them for a comparison with those at a high NaCl concentration. Absorption spectra of the m-TMPyPpoly[d(A-T)2] complex and the DNA-free m-TMPyP are depicted in Figure 2a. Upon association with poly[d(A-T)2], m-TMPyP produced hypochromism and a red shift in the absorption spectrum, particularly in the Soret absorption region, about 26% and 11 nm, respectively. The change in the absorption spectrum of the m-TMPyP-poly[d(A-T)2] complex with time is negligibly small. The magnitude of the LD spectrum in the DNA absorption region (∼260 nm) decreased immediately after mixing and was recovered to that of porphyrin-free poly[d(A-T)2] after 4 h of mixing (Figure 2b). On the other hand, that at short wavelength in the Soret region was negative: a very small positive band at long wavelength was also noticed. After time lapsed, this bisignate LD band became a large negative band, indicating that the molecular plane of porphyrin is almost parallel to the DNA base plane. The CD spectrum of m-TMPyP-poly[d(A-T)2] in the same region appeared to be strongly bisignate (Figure 2c). The magnitude of this CD band decreased and the sign reversed as time lapsed. Similar observations have been reported23,24 at a low mixing ratio and low NaCl concentration. The shape of the CD spectrum in the

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Park et al.

Figure 4. CD intensity at 427 nm of the m-porphyrin-poly[d(A-T)2] complex at the time of mixing (closed circles) and at 4 h after mixing (open circles) at various NaCl concentrations. The concentration of poly[d(A-T)2] is 100 µM in base, and that of m-porphyrin is 5 µM.

Figure 3. Absorption (a), LD (b), and CD (c) spectra of the m-TMPyP-poly[d(A-T)2] complex immediately (solid curve, curve a) and 4 h (dotted curve, curve b) after mixing in the presence of 100 mM NaCl. In panels a and b, the absorption spectrum of DNA-free m-TMPyP and the LD spectrum of porphyrin-free poly[d(A-T)2], at the same conditions, are shown (dashed curve, curve c), respectively, for comparison. The concentrations and the conditions are the same as in Figure 2.

DNA absorption region of the m-TMPyP-poly[d(A-T)2] complex showed that polynucleotides remained in the B form although some changes were observed. A negative band between 290 and 300 nm which is often reported for the porphyrinDNA system probably reflects the induced CD of the porphyrin molecule or conformation change in DNA upon porphyrin binding or both. For instance, VdOTMPyP exhibits a very strong negative CD band in this region although it retains the double-stranded B-form.28 These origins cannot be separated at this stage and therefore will not be discussed further. The effect of NaCl (100 mM) on the absorption spectrum of m-TMPyP-poly[d(A-T)2] was not significant (Figure 3a). The reduction in absorbance and red shift in the Soret band were similar to those at a low NaCl concentration. However, a unique LD spectrum was apparent for the complex both immediately and 4 h after mixing (Figure 3b). The LD signal in the DNA absorption region collapsed with a very strong positive band in the drug absorption region. As time lapsed, the positive band in the Soret region increased further and a small positive band around 260 nm was apparent. The occurrence of a collapsed LD in the DNA absorption region at the same time as the occurrence of a positive LD in the drug absorption region has not been reported before for any drug-DNA complex to our knowledge. On the other hand, the CD signal in the DNA absorption region was retained, while the magnitude of the

bisignate excitonic CD showed a tendency to increase, indicating that the secondary structure of DNA had not been altered (Figure 3c). These CD and LD properties are in dramatic contrast with those observed at a low NaCl concentration. Changes in CD and LD with NaCl Concentration. A bisignate CD spectrum with a positive maximum at 427 nm and a negative minimum at 419 nm, which has been accepted as a sign for the porphyrin stacking, was found for the m-TMPyP-poly[d(A-T)2] complex immediately after mixing at both low and high NaCl concentrations. This bisignate CD signal was retained at a high NaCl concentration. The change in the CD intensity at 427 nm was chosen to show the effect of NaCl on the CD spectrum (Figure 4). Up to a NaCl concentration of 10 mM, the CD signal at 427 nm that was read immediately after mixing decreased. The signal reached its minimum at 10 mM NaCl and then started to increase as the NaCl concentration further increased. In contrast, the CD signal at 4 h after mixing was small and negative at this wavelength: the signs of both the positive and negative signals of the bisignate CD signal were reversed. Around a NaCl concentration of 10 mM, the positive sign started to appear and increased with increasing NaCl concentration. At a high NaCl concentration (above 60 mM), the shape of the bisignate CD spectrum remained while the magnitude showed a tendency to increase. Changes in the LD intensity at various wavelengths and NaCl concentrations are depicted in Figure 5. A decrease in the magnitude of the LD intensity at 260 nm (Figure 5a) was apparent for the porphyrin-free poly[d(A-T)2] as was expected from the reduction of the repulsive interaction between the negatively charged phosphate groups, thereby resulting in an increase of DNA flexibility upon interaction with Na+ ions. At a low NaCl concentration (lower than about 10 mM), the LD magnitude of the m-TMPyP-poly[d(A-T)2] complex at the time of mixing is comparable to or larger than that in the absence of porphyrin. The magnitude tended to decrease as time lapsed. Above a NaCl concentration of 30 mM, the LD signal is much smaller compared to that in the absence of m-TMPyP. After 4 h, the magnitude decreased further to near zero or a positive signal, indicating that the orientation of DNA is very poor or nearly all DNA bases are at the magic angle relative to the flow direction. The LD signal at 427 nm (Figure 5b) immediately after mixing was small negative or bisignate at low NaCl concentrations. An increase in the negative direction was observed as time lapsed. In contrast, at a NaCl concentration above 30 mM, a positive LD signal was apparent which increased in the positive direction. The apparent positive LD

Stacking of m-TMPyP on Poly[d(A-T)2]

Figure 5. Magnitude of the LD of the m-porphyrin-poly[d(A-T)2] complex in the DNA absorption region (260 nm, panel a) and the Soret region (427 nm, panel b) measured at the time of mixing (closed circles) and at 4 h after mixing (open circles). The LD intensity of porphyrinfree poly[d(A-T)2] at 260 nm immediately after mixing, which remains the same for at least several hours, is shown as triangles in panel a.

signal in the Soret absorption band at high NaCl concentrations is correlated with a very small negative and/or positive LD signal in the DNA absorption region (at 260 nm), while a negative LD in the Soret band is accompanied by a relatively strong negative LD signal in the DNA absorption region. This odd NaCl-concentration-dependent LD signal has not been reported previously. Comparison of Spectral Properties between the m-TMPyP-Poly[d(A-T)2] and p-TMPyP-Poly[d(A-T)2] Complexes. Absorption, CD, and LD spectra of the p-TMPyP-poly[d(AT)2] complex are shown in Figure 6 for comparison, although they have already been reported.20,22,23 At a low NaCl concentration, the p-TMPyP-poly[d(A-T)2] complex is characterized by a small red shift and hypochromism in the Soret band, a negative band in both DNA and porphyrin absorption spectra, and a bisignate band in the CD spectrum, which is different from the CD signal observed for the m-TMPyP-poly[d(A-T)2] complex. At a NaCl concentration of 100 mM, no difference was observed in the absorption spectrum. A decrease in the LD magnitude, with its shape retained, may be attributed to the decrease in orientability due to the reduced repulsive interaction between the phosphate groups upon NaCl binding. In the CD spectrum, the bidignate CD spectrum becomes a positive band in the same region, indicating that p-TMPyP binds at the groove of the polynucleotide in monomeric form.13,22,23 All spectral properties of the p-TMPyP-poly[d(A-T)2] complex are in sharp contrast with those discussed in the previous sections for the m-TMPyP-poly[d(A-T)2] complex.

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Figure 6. Absorption (a), LD (b), and CD (c) spectra of the p-TMPyPpoly[d(A-T)2] complex in the presence (solid curve, curve a) and absence (dotted curve, curve b) of 100 mM NaCl immediately after mixing. In panels a and b, the absorption spectrum of DNA-free p-TMPyP and the LD spectrum of porphyrin-free poly[d(A-T)2], at the same conditions, are shown (dashed curve, curve c), respectively, for comparison. The spectral properties at 4 h after mixing were essentially unchanged. The concentration of poly[d(A-T)2] is 100 µM in the base, and that of p-porphyrin is 5 µM.

Discussion Stacking of m-TMPyP along Poly[d(A-T)2] and Abnormality of the LD Spectrum. The bisignate CD spectrum in the Soret absorption band, also called the excitonic CD spectrum, has been generally accepted as a diagnostic for porphyrin stacking along the DNA stem. Extensive and moderate selfstacking of various porphyrins with electron-rich and -deficient tentacle porphyrins, including meso-tetrakis[4-[(3-(trimethylammonio)propyl)oxy]phenyl]porphyrin, along the DNA stem have been reported.4,21,22,29 Extensive stacking favors a high [porphyrin]/[DNA] ratio and a high NaCl concentration and is characterized by a relatively low intensity conservative CD, while moderately stacked porphyrin favors low mixing ratios and a low NaCl concentration. The CD signal is more intense for moderately stacked porphyrin. On the other hand, Pasternack and co-workers reported an extensive assembly of DNA-bound trans-bis(N-methylpyridiniumyl)diphenylporphyrin and its Cu(II) complex.30,31 This species presents an induced CD signal of an unusual shape and size in the Soret absorption band, in the sense that the CD signal is bisignate but markedly nonsymmetric and its magnitude is 1-2 orders larger than that of monomeric porphyrin.11,17,18,30 Such a shape and size of the CD signal suggest an extended, electronically coupled organized array of porphyrin molecules.18,30

17110 J. Phys. Chem. B, Vol. 108, No. 44, 2004 The appearance of the m-TMPyP-poly[d(A-T)2] complex in CD and LD spectra at various NaCl concentrations can be classified into three categories. At a low NaCl concentration and immediately after mixing, a bisignate CD spectrum with its magnitude as large as its absolute values of ∆ in the 102-103 cm-1 M-1 region was apparent (Figure 2c and Figure 4). The CD spectrum in the DNA absorption region showed that DNA retained the B form in the complex. This bisignate CD species is accompanied by a small bisignate (or negative) LD in the Soret absorption region and decreased magnitude in the DNA absorption region (Figure 2b), indicating that the porphyrin plane tilts significantly with respect to the DNA base plane (near 60° relative to the flow direction). A decrease of the magnitude in the DNA absorption region is also noticed, indicating that the orientability of DNA decreases upon porphyrin stacking due either to a decrease in the repulsive interaction between phosphate groups or to a tilt of DNA base pairs. As time lapsed, the CD intensity decreased to a small bisignate CD signal with its sign reversed. In the complex, porphyrin is more parallel to the plane of the DNA base pair compared that at the time of the mixing. The orientability of poly[d(A-T)2] was almost fully recovered at 0 mM NaCl, while this recovery was less pronounced as the NaCl concentration increased to 10 mM as was reported.23,24 The last type of stacking was accompanied by a bisignate CD spectrum in the Soret absorption band with its intensity higher than that of the first type. This species favors a high NaCl concentration (at least 10 mM). The most striking observation for this species was the appearance of the LD spectrum (Figure 3b). At the time of mixing, a strong positive band in the Soret absorption region and near-zero LD magnitude in the DNA absorption region were apparent. The magnitude in the Soret region increased and a small positive signal near 260 nm appeared as time lapsed. Judging by the shape and the bandwidth, the positive band at 260 nm conceivably originated from porphyrin and not from DNA. To account for the size and bisignate nature of the induced CD signal, m-TMPyP in the complex is concluded to be assembled in some extended manner involving periodic repeats.18 However, the CD spectrum does not necessarily reflect the aggregation state. The combination of CD and resonance light scattering experiments showed that certain porphyrin derivatives can form extended assemblies on DNA templates in which DNA remained in the helical form.18,31 Our observation in this study showed that a similar extended assembly of m-TMPyP is formed in which the secondary structure of DNA is retained, as judged by CD in the DNA absorption region. However, DNA in the assembly seems to be no longer in the threadlike form: DNA conceivably is tightly compacted and not to be oriented in the flow. Effect of NaCl and Rotation of the Peripheral Pyridinium Ring on the Porphyrin Assembly. As was shown in the previous section, the type of porphyrin interaction on the DNA template can be determined by the difference in NaCl concentration. At a NaCl concentration below 10 mM the secondary structure of DNA is retained and can be oriented in the flow. However, at a NaCl concentration above 10 mM, DNA loses its threadlike property despite its unchanged B-form secondary structure. A turning point that determines those two types of porphyrin, in our condition, is 10 mM NaCl. NaCl interacts with polynucleotides, resulting in reduced repulsion between the DNA phosphate groups. This electrostatic interaction reduces the orientability of polynucleotides by increasing their flexibility (Figure 5a). In the absence of porphyrin, the LD magnitude in the DNA absorption region of poly[d(A-T)2] decreases as the

Park et al. NaCl concentration increases. The decrease reaches a plateau when the NaCl concentration reaches 10 mM. Therefore, it is conclusive that the reduction of the repulsive interaction between the phosphate groups is an important factor for the extended assembly of porphyrin. In addition to the NaCl concentration, electron deficiency/ richness and the shape of the porphyrin molecule are also important factors affecting porphyrin assembly. The observation that m-TMPyP stacks far more effectively than p-TMPyP can be explained by the repulsive interaction between the methylpyridinium rings of the other porphyrins in the assembly: in m-TMPyP, this repulsion can be minimized by the rotation of the peripheral methylpyridinium ring, providing a flexible distance between the positive charges of the porphyrin. It is also a possible reason that the m-TMPyP can form a complex more effectively than p-TMPyP as a result of the freely rotating peripheral ring. On the other hand, in the p-TMPyP molecule, the distance between the positive groups in the molecule cannot be changed although the peripheral methylpyridinium ring can rotate. Acknowledgment. This work was supported by the Korea Research Foundation (Grant No. KRF 2002-070-C00053). References and Notes (1) Fiel, R. J. J. Biomol. Struct. Dyn. 1989, 6, 1259-1274. (2) Marzilli, L. G. New J. Chem. 1990, 14, 409-420. (3) Pasternack, R. F.; Gibbs, E. J. In Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 1996; pp 367-397. (4) Marzilli, L. G.; Banville, L. D.; Zon, G.; Wilson, W. D. J. Am. Chem. Soc. 1986, 108, 4188-4192. (5) Guliaev, A. B.; Leontis, N. B. Biochemistry 1999, 38, 1542515437. (6) Lee, Y.-A.; Lee, S.; Cho, T.-S.; Kim, C.; Han, S. W.; Kim, S. K. J. Phys. Chem. B 2002, 106, 11351-11355. (7) Strickland, J. A.; Marzilli, L. G.; Wilson, W. D. Biopolymers 1990, 29, 1307-1323. (8) Kuroda, R.; Tanaka. H. J. Chem. Soc., Chem. Commun. 1994, 1575-1576. (9) Schneider, H.-J.; Wang, M. J. Org. Chem. 1994, 59, 7473-7478. (10) Sehlstedt, U.; Kim, S. K.; Carter, P.; Goodisman, J.; Vollano, J. F.; Norde´n, B.; Dabrowiak, J. C. Biochemistry 1994, 33, 417-426. (11) Pasternack, R. F.; Goldsmith, J. I.; Gibbs, E. J. Biophys. J. 1998, 75, 1024-1031. (12) Yun, B. H.; Jeon, S. H.; Cho, T.-S.; Yi, S. Y.; Sehlstedt, U.; Kim, S. K. Biophys. Chem. 1998, 70, 1-10. (13) Lee, S.; Jeon, S. H.; Kim, B.-J.; Han, S. W.; Jang, H. G.; Kim, S. K. Biophys. Chem. 2001, 92, 35-45. (14) Calvin, M. J.; Datta-Gupta, N.; Fiel, R. J. Biochem. Biophys. Res. Commun. 1982, 108, 66-73. (15) Pastenack, R. F.; Gibbs, E. J.; Villafranca, J. J. Biochemistry 1983, 22, 2406-2414. (16) Trommel, J. S.; Marzilli, L. G. Inorg. Chem. 2001, 40, 43744383. (17) Pastenack, R. F.; Ewen, S.; Rao, A.; Meyer, A. S.; Freedman, M. A.; Collings, P. J.; Frey, S. L.; Ranen, M. C.; de Paula, J. C. Inorg. Chim. Acta 2001, 317, 59-71. (18) Pasternack, R. F. Chirality 2003, 15, 329-332. (19) Lee, Y.-A.; Kim, J.-O.; Cho, T.-S.; Song, R.; Kim. S. K. J. Am. Chem. Soc. 2003, 125, 8106-8107. (20) Kim, J.-O.; Lee, Y.-A.; Yun, B. H.; Han, S. W.; Kwag, S. T.; Kim, S. K. Biophys. J. 2002, 86, 1012-1017. (21) Marzilli, L. G.; Petho¨, G.; Lin, M.; Kim, M. S.; Dixon, D. W. J. Am. Chem. Soc. 1992, 114, 7575-7577. (22) Mukundan, N. E.; Petho¨, G.; Dixon, D. W.; Kim, M. S.; Marzilli, L. G. Inorg. Chem. 1994, 33, 4676-4687. (23) Lee, Y.-A.; Lee, S.; Lee, H. M.; Lee, C.-S.; Kim, S. K. J. Biochem. 2003, 133, 343-349. (24) Lee, S.; Lee, Y.-A.; Lee, H. M.; Lee, J. Y.; Kim, D. H.; Kim, S. K. Biophys. J. 2002, 83, 371-381. (25) Ismail, M. A.; Rodger, P. M.; Rodger, A. J. Biomol. Struct. Dyn. 2000, 11, 335-348. (26) Norde´n, B.; Kubista, M.; Kurucsev, T. Q. ReV. Biophys. 1992, 25, 51-170. (27) Rodger, A.; Norde´n, B. 1 In Circular dichroism and linear dichroism; Oxford University Press: London, 1997.

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J. Phys. Chem. B, Vol. 108, No. 44, 2004 17111 (30) Gibbs, E. J.; Tinoco, I., Jr.; Maestre, M. F.; Ellinas, P. A.; Pasternack, R. F. Biochem. Biophys. Res. Commun. 1988, 157, 350-358. (31) Pastenack, R. F.; Bustamante, C.; Collings, P. J.; Giannetto, E. J.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393-5399.