Sequence-Selective Binding of Phenazinium Dyes Phenosafranin and

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Sequence-Selective Binding of Phenazinium Dyes Phenosafranin and Safranin O to Guanine-Cytosine Deoxyribopolynucleotides: Spectroscopic and Thermodynamic Studies Ishita Saha, Maidul Hossain, and Gopinatha Suresh Kumar* Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology (CSIR), Kolkata 700 032, India ReceiVed: July 13, 2010; ReVised Manuscript ReceiVed: October 4, 2010

The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 105 M-1, but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA) · poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O. Introduction Understanding the base sequence selectivity of interaction of small aromatic molecules that essentially bind to DNA by intercalation is a fundamental aspect in the rational design of gene-specific binders that are effective in the therapy of human diseases. This has been the subject matter of a large number of investigations in the past four decades or so.1 The two kinds of major DNA binding modes of small molecules involve minor groove binding and intercalation. Over the past decades, there has been extensive progress in understanding the minor groove recognition of B-DNA by small molecules from the pioneering work of Dervan and Lown through a series of designed molecules1h-j But, in spite of these success stories with groove binding molecules, development of highly sequence specific molecules targeted to DNA remains still challenging. In particular, effective intercalating chemotherapeutic agents targeted to specific sequences remain still a highly investigated area. More detailed study and careful analysis are required to elucidate the molecular aspects in terms of the structural elements and the thermodynamics of the interaction of the binding as a function of base sequence and selectivity. Phenosafranin (PSF, 3,7-diamino-5-phenylphenazinium chloride) and safranin O (SO, 3,7-diamino-2,8-dimethyl-5-phenylphenazinium chloride) (Chart 1) are the two most important cationic dyes of the phenazinium group of compounds. They both have planar conjugated rigid structure but differ in methyl substitution level. Phenazinium dyes have extensive applications in semiconductors, as energy sensitizers, in probing microheterogeneous environments, and in many biological applications * Corresponding author. Address for correspondence: Dr. G. Suresh Kumar, Ph.D., Scientist, Biophysical Chemistry Laboratory, Indian Institute of Chemical Biology (CSIR, Govt. of India), 4, Raja S. C. Mullick Road, Jadavpur, Kolkata 700 032, India. Phone: +91 33 2472 4049, 2499 5723. Fax: +91 33 2472 3967. E-mail: [email protected], [email protected].

CHART 1: Chemical Structures of (A) Phenosafranin (PSF) and (B) Safranin O (SO)

in photochemistry, DNA determination, etc.2 The DNA interaction of these dyes has been known but limited to very few reports with natural DNAs.3-5 Smekal and co-workers reported the DNA binding of phenosafranin3 and proposed both intercalation and outside binding modes. Studies in our laboratory4 and those by Chattopadhyay and co-workers5 on the binding of these dyes with natural DNAs showed intercalation mode of binding and an overall guanine-cytosine base preference. But the sequence selectivity of interaction of phenazinium dyes and the related energetics of the interaction, essential for drug development, remained incompletely described. To provide better insight into these two aspects, and augment the existing data with natural DNAs, we studied the interaction of PSF and

10.1021/jp1064598  2010 American Chemical Society Published on Web 10/27/2010

Phenazinium Dyes-Polynucleotide Complexation

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SO with four sequence-specific synthetic polynucleotides using various spectroscopic techniques, competition dialysis assay, and calorimetry experiments. Here, we present a complete structural and thermodynamic profile of the interaction of these dyes to these polynucleotides that enabled an unequivocal elucidation of the sequence selectivity of the binding of these dyes. Experimental Methods Materials. The polynucleotides, poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), poly(dA-dT) · poly(dA-dT), and poly(dA) · poly(dT) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). The samples were made to uniform size of about 280 ( 50 base pairs by sonication in a Labsonic sonicator (B Brown, Germany) using a needle probe of 4 mm diameter. After sonication, the samples were dialyzed against the experimental buffer. The sonicated samples were estimated to have a molecular weight of around 2 × 105 Da by viscosity determinations and is confirmed to be linear rodlike DNA samples ideal for intercalation studies.6 The concentration of the polynucleotides were determined by applying the molar extinction coefficients expressed in base pairs.7 Phenosafranin and safranin O (hereafter dyes), obtained from Sigma-Aldrich, were used without further purification. Dye solutions were freshly prepared in the experimental buffer and kept protected wrapped in aluminum foil. Concentrations were determined using molar extinction coefficients (ε) reported in the literature.4,5 All experiments were conducted in 20 mM sodium cacodylate buffer, pH 7.0, prepared in deionized and triple distilled water. Buffer solutions were filtered through Millipore filters of 0.45 µM (Millipore India Pvt. Ltd., Bangalore, India). Binding by Spectroscopic Measurements. Measurements were carried out at 20 °C. Binding was monitored spectrophotometrically or fluorimetrically in the dye absorption or emission regions, respectively, after addition of scalar amounts of the polynucleotides into a freshly prepared dye solution as described previously.8 Spectrophotometric and fluorimetric measurements were made with a Jasco V-660 unit and a Shimadzu RF5301 PC spectrofluorometer in thermostated quartz cells. The spectral changes observed in absorption and fluorescence titrations were utilzed to construct Scatchard plots of r/Cf versus r. Nonlinear Scatchard plots obtained were analyzed further by a nonlinear regression methodology, viz., the neighbor exclusion model of McGhee-von Hippel9

r/Cf ) Ki(1 - nr)[(1 - nr)/{1 - (n - 1)r}](n-1)

(1) where r is the molar ratio of the bound dye to polynucleotide, Cf is the free dye concentration, Ki is the intrinsic binding constant to an isolated binding site, and n is the exclusion parameter. All the binding data were analyzed using Origin 7.0 software (Microcal LLC., Northampton, MA) to determine the best-fit parameters of Ki and n. Fluorescence quenching experiments with the anionic quencher, K4[Fe(CN)6], were performed using published procedures.10 Helix Melting Studies. Ultraviolet melting profiles of polynucleotides and polynucleotide-dye complexes were measured on a Shimadzu Pharmaspec 1700 spectrophotometer (Shimadzu Corp., Kyoto, Japan) equipped with the Peltier-controlled eightchambered quartz cuvette of 1 cm path length.11 Experiments were generally conducted at a concentration of 20.0 × 10-6 M base pairs as a function of different ratios of polynucleotide-dye complex. Melting temperature (Tm) was taken as the temperature

of half-dissociation of the polynucleotide duplex and was obtained from the maximum of the first derivative dA/dT plots (where A is the absorbance and T is the temperature). Circular Dichroic (CD) Study. The CD spectra were acquired on a Jasco model J815 spectropolarimeter (Jasco International Co. Ltd., Tokyo, Japan) equipped with a temperature controller (Jasco model PFD 425 L/15) interfaced with a HP PC at 20 ( 0.5 °C with a 1 cm path length quartz cuvette.8 A scan speed of 50 nm/min with a response time of 1 s was used for scanning. The spectra from 400 to 210 and 600 to 300 nms were averaged over five scans. A buffer baseline scan was subtracted from the average scan for each sample. The titration experiments were performed at 20 °C. The molar ellipticity values [θ] (deg cm2 dmol-1) are calculated from the equation

[θ] ) [θ]obs /10lC

(2)

where [θ]obs is the observed ellipticity (millidegrees), C is the molar concentration, and l is the optical path length of the cuvette (cm). The expressed molar ellipticity is in terms of either DNA base pairs (210-400 nm) or per bound dye (300-600 nm). The data processing and plotting were performed with Origin 7 software. Competition Dialysis Assay. This was carried out generally following the methods developed by Chaires and co-workers.12,13 A 0.5 mL solution of each of the polynucleotide solution (at a concentration of 75 µM in base pair unit) in separate 0.5 mL Slide-A-Lyzer minidialysis units (Pierce Chemical Co., IL,) were dialyzed against a 1 µM dye solution for 24 h at 20 ( 0.5 °C. The apparent binding (Kapp) constants were calculated from the polynucleotide-bound dye concentrations.13 Differential Scanning Calorimetry. Excess heat capacities as a function of temperature were measured on a VP-differential scanning calorimeter (DSC; MicroCal LLC, MA). Experiments were conducted at a polynucleotide concentration of 40.0 × 10-6 M. Scans were conducted between 30 and 120 °C at a rate of 60 °C per hour. Baseline scans obtained with buffer in both the calorimeter cells were subtracted from the sample experimental thermograms. The DSC thermograms were analyzed using the Origin 7.0 software package14 to determine the calorimetric transition enthalpy (∆Hcal). The calorimetrically determined enthalpy is model-independent and not related to the nature of the transition. The temperature at which the excess heat capacity is maximum defines the transition temperature (Tm). The model-dependent van’t Hoff enthalpy (∆Hv) was obtained by shape analysis of the calorimetric data and the cooperativity factor was obtained from the ratio of the calorimetric and van’t Hoff enthalpy. Isothermal Titration Calorimetry. Isothermal titration calorimetry experiments were carried out in a VP-ITC microcalorimeter (ITC; MicroCal LLC). Instrument control, data acquisition, and analysis were performed using the dedicated Origin 7.0 software following methods described earlier.7,8 Aliquots of degassed dye solutions were injected from the rotating syringe (stirring speed 290 rpm) into the isothermal sample chamber containing the respective polynucleotide (1.4235 mL) solution. The reference cell contained distilled water. The heat liberated or absorbed with each injection of the dye aliquot is observed as a peak that corresponds to the power required to keep the sample and reference solutions at identical temperature. The peaks produced over the entire course of the titration are converted to heat output per injection by integration. The area under each heat burst curve was determined by integration using the Origin software. Control experiments were carried out to

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Figure 1. Representative absorption spectra of (A) free PSF (3 µM) treated with 0, 12, 24, 30, 36, 42, 48, 54, 60, and 63 µM (curves 1-10) of poly(dG-dC) · poly(dG-dC); (B) free SO (4 µM) treated with 0, 20, 40, 48, 56, 64, 72, 80, 88, and 96 µM of poly(dG-dC) · poly(dG-dC) (curves 1-10). Representative steady-state fluorescence emission spectra of (C) PSF (0.6 µM) treated with 0, 3, 4.8, 7.2, 9.6, 13.2, 14.4, 16.8, 18, and 19.2 µM (curves 1-10) of poly(dG-dC) · poly(dG-dC), and (D) SO (0.6 µM) treated with 0, 3.6, 4.8, 7.2, 9.6, 12, 14.4, 18, 21.6, and 25.2 µM (curves 1-10) of poly(dG-dC) · poly(dG-dC). Excitation wavelength for PSF and SO was 520 nm. All experiments were performed at 20 °C in 20 mM sodium cacodylate buffer of pH 7.0.

determine the heat contribution from dilution arising from buffer into polynucleotide and dye into buffer. The resulting corrected injection heats were plotted as a function of the molar ratio. Experimental data were fitted using a nonlinear least-squares minimization algorithm to one site binding theoretical curves to provide the binding affinity (Ka), the binding stoichiometry (N), and the enthalpy of binding (∆H). The binding site size was restricted during the fitting process to values close to that obtained from the spectroscopic data. The binding free energy (∆G) and the entropic contribution (T∆S) were deduced from the standard relationships ∆G ) -RT ln Ka (R ) 1.9872 cal mol-1 K-1, T ) 298 K) and ∆G ) ∆H - T∆S. Results and Discussion Spectroscopic Experiments: Elucidation of the Binding Parameters. Upon titration with polynucleotide solutions, PSF and SO exhibited dramatic changes in their spectroscopic properties. In particular, the characteristic visible absorption spectra underwent bathochromic and hypochromic shifts generating an isosbestic point. Isosbestic points observed in the titration indicated the presence of two distinct spectroscopic species, viz., the bound and free form, in equilibrium. Representative absorption titration profiles of PSF and SO with poly(dG-dC) · poly(dG-dC) is presented in Figure 1A,B. The fluorescence emission of PSF and SO was progressively quenched in the presence of the GC polynucleotides while with the two AT polynucleotides the fluorescence enhanced. Representative fluorescence titration profiles of PSF with poly(dGdC) · poly(dG-dC) and poly(dG) · poly(dC) are presented in Figure 1C,D. The enhancement of the fluorescence of these dyes on complexation with the AT polynucleotides may be considered due to the shielding from transient interactions like H-bonding with water molecules. On the other hand, the quenching on

Saha et al.

Figure 2. Respective Scatchard plots of (A) PSF-poly(dG-dC) · poly(dGdC) (crossed squares), PSF-poly(dG) · poly(dC) (b), PSF-poly(dAdT) · poly(dA-dT) (2), and PSF-poly(dA) · poly(dT) ([) and (B) SO-poly(dG-dC) · poly(dG-dC) (X), SO-poly(dG) · poly(dC) (O), SO-poly(dA-dT) · poly(dA-dT) (4), and SO-poly(dA) · poly(dT) (]) bindings at 20 mM sodium cacodylate buffer, pH 7.0. The points in the figure represent the actual data points, and the solid lines represent the best fit to McGhee-von Hippel equation.8 The experimental points are the average of four determinations.

complexation with the GC polynucleotides may involve electron transfer from guanine to the excited state of the dyes15 leading to transient guanine cations and dye anion radical formation as suggested for quinacrine and methylene blue.16,17 The Scatchard plots (Figure 2) constructed from the spectroscopic data revealed that the binding of both dyes to all the four polynucleotides investigated was noncooperative. Analysis of the Scatchard plots by McGhee-von Hippel equation yielded the binding parameters reported in Table 1. The binding affinity (Ki) of PSF and SO to poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), and poly(dA-dT) · poly(dA-dT) were of the order of 105 M-1, PSF having higher affinity compared to SO in each case. The binding affinity values of PSF and SO to poly(dA) · poly(dT) were the lowest and with SO it was of the order of 104 M-1. The numbers of excluded sites (Table 1) for the binding of PSF and SO to poly(dG-dC) · poly(dG-dC) and poly(dG) · poly(dC) were lower and in the range 2-3, reiterating the binding to obey neighbor exclusion principle9 and the higher binding density of PSF and SO on the GC polynucleotides. Intrinsic and Induced Circular Dichroic Study. Circular dichroism is an informative technique that can provide information about the binding mode.1g The intercalation of PSF and SO to these duplex polynucleotides induced changes in the intrinsic CD of the polynucleotides and also generated induced CD in the dye absorption in the visible region. Note that the dyes are optically inactive and do not have any CD spectra. Comparative circular dichroic changes in the four polynucleotides on binding of PSF and SO at various D/P (dye/ polynucleotide base pair molar ratio) values were recorded in the 210-400 nm region. The data are presented in Figure 3. The binding results in terms of the enhancement in the ellipticity of the long wavelength positive and negative bands reflecting lengthening of the helix and weakening of the base stacking interactions due to intercalation4,5,18 and the change was maximum for poly(dG-dC) · poly(dG-dC) followed by poly(dG) ·



TABLE 1: Binding Parameters for the Complexation of PSF and SO with Polynucleotides Obtained from Scatchard Analysis of the Absorbance and Fluorescence Titration Dataa absorbance

fluorescence

PSF polynucleotide poly(dG-dC) · poly(dG-dC) poly(dG) · poly(dC) poly(dA-dT) · poly(dA-dT) poly(dA) · poly(dT)

Ki × 10

-5

-1

(M )

4.86 ( 0.20 3.44 ( 0.30 2.62 ( 0.23 0.92 ( 0.11

SO n 2.31 2.62 2.86 3.31

Ki × 10

-5

PSF

-1

(M )

3.27 ( 0.18 2.02 ( 0.15 1.57 ( 0.12 0.55 ( 0.08

n 2.62 2.94 3.91 4.19

Ki × 10

-5

-1

(M )

4.91 ( 0.18 3.50 ( 0.16 2.83 ( 0.09 1.01 ( 0.07

SO n 2.28 2.52 2.67 3.40

Ki × 10

-5

(M-1)

3.43 ( 0.11 2.39 ( 0.10 1.80 ( 0.07 0.76 ( 0.05

n 2.51 3.01 3.82 4.30

a Average of four determinations. Binding constants (Ki) and the number of occluded sites (n) (in base pairs) refer to solution conditions of 20 mM cacodylate buffer, pH 7.0 at 20 °C.

Figure 3. Intrinsic circular dichroic spectra (upper panels) of (A) 30 µM poly(dG-dC) · poly(dG-dC) treated with 0, 3, 6, 12, 15, and 18 µM of PSF, (B) 30 µM of poly(dG) · poly(dC) treated with 0, 3, 6, 15, 18, and 21 µM of PSF, (C) 30 µM of poly(dA-dT) · poly(dA-dT) treated with 0, 3, 9, 15, 24, and 27 µM of PSF, and (D) 30 µM of poly(dA) · poly(dT) treated with 0, 9, 15, 24, 27, and 30 µM of PSF. (Lower panels) (E) 30 µM poly(dG-dC) · poly(dG-dC) treated with 0, 1.5, 6, 12, 18, and 21 µM of SO, (F) 30 µM poly(dG) · poly(dC) treated with 0, 6, 12, 18, 21, and 22.5 µM of SO, (G) 30 µM poly(dA-dT) · poly(dA-dT) treated with 0, 6, 15, 21, 27, and 33 µM of SO, and (H) 30 µM poly(dA) · poly(dT) treated with 0, 9, 15, 28.5, 30, and 39 µM of SO. All the experiments were performed at 20 °C in 20 mM cacodylate buffer of pH 7.0.

Figure 4. Induced circular dichroic spectra of PSF (30 µM) treated with (upper panels) (A) 0, 12, 24, 30, 60, and 75 µM of poly(dG-dC) · poly(dGdC), (B) 0, 12, 24, 60, 75, and 105 µM of poly(dG) · poly(dC), (C) 0, 30, 45, 105, 114, and 120, µM of (dA-dT) · poly(dA-dT), and (D) 0, 42, 60, 90, 120, and 135 µM of poly(dA) · poly(dT). (Lower panels) Induced circular dichroic spectra of SO (30 µM) treated with (E) 0, 12, 36, 60, 75, and 90 µM of poly(dG-dC) · poly(dG-dC), (F) 0, 27, 45, 75, 90, and 115 µM of poly(dG) · poly(dC), (G) 0, 30, 66, 90, 120, and 132 µM of poly(dAdT) · poly(dA-dT), and (H) 0, 30, 60, 90, 126, and 150 µM of poly(dA) · poly(dT). All the experiments were performed at 20 °C in 20 mM cacodylate buffer of pH 7.0.

poly(dC). The changes were smaller in poly(dA-dT) · poly(dAdT) and only marginal with poly(dA) · poly(dT). The changes were essentially more with PSF compared to SO. The induced CD spectra appeared in the dye absorption region (400-600 nm) for the bound dye molecules (Figure 4). With the two GC polynucleotides, the induced CD spectra was bisignate. It is significant to note that the negative and positive bands for the alternating GC polynucleotide centered around 500 and 550 nm, respectively, had similar ellipticity values while

the negative band with the GC homopolynucleotide is much smaller with compared to the positive band. The negative CD band at the lower wavelength and the positive CD band at the higher wavelength are typical of chromophores that have a righthanded orientation for dyes bound to B-DNA structure19 as per the predictions of exciton coupling theory. The negative band is hardly seen in the induced CD spectra of the dye-alternating AT polynucleotide complexes. Further, the ellipticity values of the induced CD bands were larger with poly(dG-dC) · poly(dG-

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Figure 5. Thermal melting profiles (upper panels) of (A) poly(dG) · poly(dC) (b), (B) poly(dA-dT) · poly(dA-dT) (2), (C) poly(dA) · poly(dT) (1), PSF-DNA complex (9), SO-DNA complex (0), DSC thermograms (lower panels) of (D) poly(dG) · poly(dC), (E) poly(dA-dT) · poly(dA-dT), (F) poly(dA) · poly(dT) (—), and DNA-PSF complex (---), DNA-SO complex (...).

dC) and small with poly(dG) · poly(dC) and smallest with poly(dA-dT) · poly(dA-dT). The induced CD signal of both PSF and SO with poly(dA) · poly(dT) was very weak and could not be distinguished from noise. Thus, intrinsic and induced CD results also confirm the sequence selectivity of these dyes to the alternating GC sequences and the preference of PSF over SO in conformity with the binding results from spectroscopic experiments. The intercalative mode of binding of these dyes has been independently verified by ferrocyanide quenching and viscosity (vide infra). Verification of Intercalation from K4[Fe(CN)6] Fluorescence Quenching and Viscosity Measurements. A method of investigating the mode of binding is provided by fluorescence quenching experiments.20 In the complex, molecules that are free or bound weakly on the surface of the helix are readily available to the quencher, while those that are intercalated are not. If the quencher is anionic in nature, the electrostatic barrier due to the negative charges on the phosphate groups at the helix surface may restrict its penetration into the interior of the helix. Hence very little or no quenching should be observed in presence of an anionic quencher, if the binding involves intercalation and consequently the magnitude of the Stern-Volmer quenching constant (Ksv) of dyes that are bound inside will be lower than that of the free molecules.21 The Stern-Volmer plots of the complexation revealed (Supporting Information, Figure S1) that the Ksv values for free PSF and SO were 27.42 and 30.81 L mol-1, respectively. For the PSF molecules bound with poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), poly(dAdT) · poly(dA-dT), and poly(dA) · poly(dT), Ksv values were 8.72, 12.43, 17.38, and 24.32 L mol-1 and the same for SO were 11.39, 16.37, 23.15, and 28.55 L mol-1. This result indicates that the accessibility of the quencher to the bound dye molecules with the GC polynucleotides are practically restricted to a much higher extent compared to the AT polynucleotides. The protected and sequestered away from the solvent dye molecules suggest most likely intercalative binding as reported with natural DNAs also.4,5 The intercalative binding of PSF and SO to the polynucleotides was further tested by viscosity measurements. Viscometric technique is a well-established and reliable hydrodynamic

method22 for investigating the extension of double-stranded DNA helix on intercalation. The hypothesis of Lerman23 proposed that the viscosity of a rodlike nucleic acid increases upon complexation with an intercalator. The viscosity of the polynucleotide solutions in the presence of increasing concentration of the dyes was measured, and the changes in relative viscosities with varying D/P ratios of the dyes were estimated (Supporting Information, Figure S2). The viscosity change was found to be more pronounced for polynucleotide complexes with PSF compared to SO. Viscosity results are usually expressed as relative length enhancement (L/L0) estimated with respect to a standard value (β) of 1.0 for a true intercalator corresponding to a length enhancement of 0.34 nm per bound dye. The β values for PSF and SO binding to poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), poly(dA-dT) · poly(dA-dT), and poly(dA) · poly(dT) were 0.87, 0.81 0.71, and 0.66, respectively. For SO complexes with poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), poly(dAdT) · poly(dA-dT), and poly(dA) · poly(dT), the β values were 0.84, 0.78 0.69, and 0.63, respectively, again suggesting most likely a better intercalative binding with the GC polynucleotides compared to the AT polynucleotides. Fluorescence quenching and viscometry are complementary techniques providing good evidence for the binding mode of these molecules to the sequence of base pairs. Thus, a true intercalation scenario may be envisaged for PSF and SO binding to the GC polynucleotides whereas partial intercalation may be assigned to binding to the AT polynucleotides. Helix Melting from Absorbance and Differential Scanning Calorimetry. The ability of PSF and SO to enhance the thermal stability was studied through absorbance melting experiments and by differential scanning calorimetry. The melting temperatures of poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), poly(dAdT) · poly(dA-dT), and poly(dA) · poly(dT) were 98.0, 88.0, 48.0, and 54.0 °C, respectively, under the buffer conditions used here. The binding of the dyes strongly stabilized all the polynucleotides. No finite stabilization data was obtained with poly(dGdC) · poly(dG-dC)-dye complexes because the Tm values at all D/P values were >110 °C (Figure 5A). Under saturating conditions, ∆Tm values of about 18.0, 17.0, and 10.0 °C were obtained with PSF and 16.0, 14.0, and 8.0 °C with SO for



TABLE 2: Thermal Melting Data and Binding Constants from Optical Melting Data at Saturating Concentrations of PSF and SO with Polynucleotidesa system DNA

dye

Tm (°C) (optical melting)

Tm (°C) (DSC)

∆Tm

poly(dG-dC) · poly(dG-dC)

PSF SO PSF SO PSF SO PSF SO

98.5 nd nd 87.0 nd nd 48.6 65.0 62.5 54.6 66.5 64.6

99.1 nd nd 88.4 106.3 104.7 47.2 64.3 61.8 55.4 65.2 63.1

nd nd 17.9 ( 0.9 16.3 ( 0.7 17.0 ( 0.5 14.6 ( 0.9 9.8 7.7

poly(dG) · poly(dC) poly(dA-dT) · poly(dA-dT) poly(dA) · poly(dT)

KTm (M-1) × 10-5

Kobs (M-1) × 10-5

nd nd

nd nd

2.53 ( 0.12 2.25 ( 0.12 2.45 ( 0.06 0.93 ( 0.12 nd nd

4.90 ( 0.12 4.35 ( 0.12 3.96 ( 0.06 1.37 ( 0.12 nd nd

a Melting stabilization of DNA duplexes in the presence of saturating amounts of the dyes. The melting profiles were recorded at the wavelength maxima of the polynucleotides. The data are average of four determinations. KTm is the binding constant at the melting temperature, and Kobs is the binding constant calculated for 20 °C. nd ) not determined.

Figure 6. Results of competition dialysis assay of PSF and SO binding to various polynucleotides at 20 °C in 20 mM cacodylate buffer, pH 7.0. The concentration of PSF and SO bound each polynucleotide sample is depicted as a bar graph. The data given are average of three independent experiments under identical conditions run concurrently. Values at the right are the apparent binding constants for the polynucleotides calculated as described by Chaires.12

poly(dG) · poly(dC), poly(dA-dT) · poly(dA-dT), and poly(dA) · poly(dT). The melting profiles are presented in Figure 5A-C. DSC studies revealed cooperative reversible melting profiles (∆Hcal ) ∆Hv) for the native polynucleotides (Figure 5D-F) and revealed similar stabilization values as observed from optical melting studies. Melting temperature data were used to calculate the binding constants (Kobs) of the association of PSF and SO to poly(dG) · poly(dC) and poly(dA-dT) · poly(dA-dT) using the procedure of Crothers.24 The values calculated from these data for 20 °C are presented in Table 2. The values of Kobs are of the same order and close to the values of Ki obtained from spectroscopic studies. In the case of poly(dA) · poly(dT), Kobs could not be calculated by this procedure as the ∆H value could not be deduced from the anomalous ITC data. Guanine-Cytosine Sequence Selectivity of PSF and SO from Dialysis Assay. The results of competition dialysis assay are presented in Figure 6 as bar graphs showing the concentration of PSF and SO bound to each of the polynucleotide. The binding in terms of accumulation of the dyes was found to be highest with poly(dG-dC) · poly(dG-dC), and varied as poly(dG) · poly(dC)>poly(dA-dT) · poly(dA-dT)> poly(dA) · poly(dT) in that order for both dyes. The striking result that emerged from this experiment was the pronounced binding of both PSF and SO to poly(dG-dC) · poly(dG-dC). Poly(dG) · poly(dC) represents the

next most preferred sequence followed by poly(dA-dT) · poly(dAdT). Poly(dA) · poly(dT) has the least and much lower binding preference compared to other polynucleotides for both the dyes. Further, it is also clear that, between the two dyes, PSF has a higher preference for the polynucleotides. The higher preference of PSF over SO has been reported from studies with natural DNAs also.4b The apparent binding affinities (Kapp) have been calculated from the concentrations of the bound dyes (Figure 6). These results unequivocally suggest that the binding affinities of PSF was higher than those of SO to the polynucleotides, the binding was highest with poly(dG-dC) · poly(dG-dC), and the affinities of both the dyes varied in the same order as the results from the spectroscopic and helix melting experiments. Energetics of the Binding from Isothermal Titration Calorimetry Results. After the guanine-cytosine sequence selectivity of PSF and SO was established, the energetics of the interaction was elucidated by calorimetry. Here we present detailed calorimetric analysis of the binding of PSF and SO to the four polynucleotides. In the top panels of Figures 7 and 8 are presented the primary data from the titration of PSF and SO into various polynucleotide solutions at 20 °C. The binding was characterized by exothermic heats in each case and the heat evolved with poly(dG-dC) · poly(dG-dC) was higher than that produced with the others. It can be seen that PSF binding generated more heat than SO with each of the polynucleotides. The ITC pattern of the dyes with poly(dA) · poly(dT) showed an initial decrease and then an increase followed by further increase in the heat evolution. Since the integrated heat data in the ITC profiles of all other polynucleotides showed only one binding event (lower panel of Figures 7 and 8), they were fitted to a single set of identical sites model. This was also based on the results from single binding mode revealed from Job plot analysis25 that yielded a well-defined 1:1 dye-polynucleotide stoichiometry (Supporting Information, Figure S3). The data from ITC experiments are presented in Table 3. It can be seen that the binding affinity values obtained from ITC data are of the order of 105 M-1 and follow exactly the same trend as observed from spectroscopic studies, being highest for poly(dGdC) · poly(dG-dC) and varying in the order poly(dG-dC) · poly(dGdC) > poly(dG) · poly(dC) > poly(dA-dT) · poly(dA-dT) for both the dyes. The values for PSF were significantly higher than those of SO. The binding of the dyes to each of the polynucleotide was driven by higher positive entropy changes and smaller negative enthalpy changes. Particularly noteworthy was the data of PSF with poly(dA-dT) · poly(dA-dT) where the binding was

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Figure 7. Representative ITC profiles for the titration of (A) poly(dG-dC) · poly(dG-dC), (B) poly(dG) · poly(dC), (C) poly(dA-dT) · poly(dA-dT), and (D) poly(dA) · poly(dT) with PSF at 20 °C in 20 mM cacodylate buffer of pH 7.0. The top panels represent the raw data for the sequential injection of dye into DNA polynucleotides, and the bottom panels show the integrated data after correction of heat of dilution against the molar ratio of dye/DNA polynucleotide. The data (filled squares) were fitted to a one-site model, and the solid lines represent the best-fit data.

Figure 8. Representative ITC profiles for the titration of (A) poly(dG-dC) · poly(dG-dC), (B) poly(dG) · poly(dC), (C) poly(dA-dT) · poly(dA-dT), and (D) poly(dA) · poly(dT) with SO at 20 °C in 20 mM cacodylate buffer of pH 7.0. The top panels represent the raw data for the sequential injection of dye into DNA, and the bottom panels show the integrated data after correction of heat of dilution against the molar ratio of dye/DNA polynucleotide. The data (filled squares) were fitted to a one-site model, and the solid lines represent the best-fit data.

TABLE 3: Isothermal Calorimetric Data for the Binding of PSF and SO to Polynucleotidesa DNAs

dye

T (K)

Ka × 10-5 (M-1)

n

∆G (kcal/mol)

∆H (kcal/mol)

T∆S (kcal/mol)

poly(dG-dC) · poly(dG-dC)

PSF

283 293 303 283 293 303 283 293 303 283 293 303 283 293 303 283 293 303

8.91 ( 0.91 4.30 ( 0.90 2.39 ( 0.42 5.23 ( 0.70 3.16 ( 0.61 2.02 ( 0.57 5.13 ( 0.50 3.74 ( 0.17 2.06 ( 0.12 3.26 ( 0.15 2.24 ( 0.10 1.68 ( 0.09 4.14 ( 0.40 2.45 ( 0.45 1.38 ( 0.39 2.87 ( 0.40 1.78 ( 0.33 1.01 ( 0.43

2.15 2.30 2.53 2.33 2.62 2.75 2.16 2.57 2.69 3.27 3.48 3.84 2.21 2.58 2.78 3.58 4.16 4.48

-7.69 -7.54 -7.47 -7.40 -7.36 -7.35 -7.39 -7.48 -7.36 -7.14 -7.17 -7.22 -7.27 -7.23 -7.11 -7.07 -7.05 -6.94

-1.78 -3.15 -4.38 -1.17 -2.09 -3.20 -1.73 -2.62 -3.57 -0.91 -1.73 -2.53 -0.88 -2.05 -3.08 -0.53 -1.48 -2.40

5.91 4.39 3.09 6.23 5.27 4.15 5.66 4.86 3.79 6.23 5.44 4.69 6.39 5.18 4.03 6.54 5.57 4.54

SO poly(dG) · poly(dC)

PSF SO

poly(dA-dT) · poly(dA-dT)

PSF SO

∆Cp (cal/(mol K))

∆Ghyd (kcal/mol)

-130

-10.4

-101

-8.1

-92.3

-7.4

-80.7

-6.5

-110.0

-8.8

-93.7

-7.5

a All the data in this table are derived from ITC experiments conducted in 20 mM cacodylate buffer, [Na+], pH 7.0, and are average of three determinations. Ka and ∆H values were determined from ITC profiles fitting to Origin 7 software as described in the text. The values of ∆G and T∆S were determined using the equations ∆G ) -RT ln Ka, and T∆S ) ∆H - ∆G. All the ITC profiles were fit to a model of single binding sites. n is the reciprocal of N.

highly entropy dominated. The strong positive entropy term is suggestive of the disruption and release of bound water molecules on intercalation of PSF and SO into the helix of this

polynucleotide. Similar result was proposed for the binding of these dyes to AT-rich natural DNA also.4b The overall binding affinity and the binding site size values obtained from ITC



analysis are in excellent agreement with the affinity values and the stoichiometry values obtained from spectroscopy. These values are also comparable to those evaluated from the thermal melting data. Thus, the ITC data also clearly suggest the GC selectivity of both the dyes on the one hand and the stronger binding of PSF over SO on the other. It is perhaps pertinent to comment about the anomalous thermogram of complexation with poly(dA) · poly(dT). In poly(dA) · poly(dT) (Figures 7D and 8D), the thermogram revealed unusual binding that could not be fitted to any protocol. The enthalpy initially increased, then decreased, and finally increased with both PSF and SO. These results suggest the existence of more than one type of binding that was not discerned in the spectroscopic experiments. We investigated the role of salt concentration in the unusual ITC of poly(dA) · poly(dT) by performing titration at 100 mM [Na+] but observed that the binding was remarkably reduced at this [Na+] concentration showing only background heats. This polynucleotide has a different structure from the canonical B-form.26,27 Poly(dA-dT) · poly(dA-dT) has a classical B-form structure while poly(dA) · poly(dT) has a nonstandard structure with a distinctly narrow minor groove and wider major groove than the B-form structure that results in a rigid bent structure.26 The rigidity in the structure of the homopolynucleotide also arises from the high propeller twist of the base pairs. Also, this polynucleotide has a unique spine of energetically favorable water structure in the minor groove. Many drug binding studies have reported anomalous behavior with several intercalators and groove binders.28-34 Herrera and Chaires29 reported a premelting conformational transition in poly(dA) · poly(dT) coupled to daunomycin binding. Anomalous behavior in the volumetric parameters on ethidium binding has also been reported with this polynucleotide.33 In the absence of high-resolution structural data, the exact reasons for the anomalous behavior of this polynucleotide on dye binding remains unclear. Dependence of Binding on Ionic Strength. The phenazinium dyes are cationic molecules with single positive charge on the exocyclic nitrogen atom. DNA condenses counterions to its surfaces to screen the polyanionic charge on the phosphodiester backbone. Thus, electrostatic interaction is thought to be a predominant force in the binding process. To provide insight into the role of electrostatic interaction, ITC binding assays at varying concentrations of Na+ in conjugation with van’t Hoff analysis were performed for poly(dG-dC) · poly(dG-dC) and poly(dG) · poly(dC). A relation between binding constant and sodium ion concentration has been derived by Record et al.35

δ log(Ka)/δ log([Na+]) ) -Zφ

(3)

where Z is the apparent charge on the bound dye and φ is the proportion of counterions (sodium ions) bound with each DNA phosphate group. The binding affinities were tested at three salt concentrations 20, 50, and 100 mM [Na+]. The log of Ka values derived from ITC experiments was then plotted against log of the molar Na+ concentration (Supporting Information, Figure S4A,B). According to counterion condensation theory, such a representation should give a linear plot with the slope of the best linear least-squares fit proportional to the number of Na+ ions released. The slopes were found to be -0.90 and -0.87 for PSF and -0.86 and -0.79 for SO binding to poly(dGdC) · poly(dG-dC) and poly(dG) · poly(dC). These values correspond quite well to one positive charge carried by PSF and SO at neutral pH (Chart 1). Knowledge of the binding affinity and the enthalpy of binding allows the construction of a complete thermodynamic profile

TABLE 4: Partitioning of the Binding Free Energy of the Complexation of the Binding of PSF and SO to Poly(dG-dC) · poly(dG-dC) and Poly(dG) · poly(dC) at 20 mM [Na+]a

complex poly(dG-dC) · poly(dG-dC) PSF SO poly(dG) · poly(dc) PSF SO

-Zφ

∆G (kcal/ mol)

∆Gt (kcal/ (mol K))

∆Gpe (kcal/ mol)

0.90 0.87

-7.55 -7.37

-5.50 -5.39

-2.05 -1.98

0.86 0.79

-7.47 -7.16

-5.51 -5.37

-1.96 -1.79

a All the data in this table are derived from ITC experiments fit to a model of single binding site. The values of ∆G were determined using the equations ∆G ) -RT ln Ka.

of interaction for these dyes. From the dependence of Ka on [Na+], the observed binding free energy (∆Gobs) can be partitioned between the nonpolyelectrolytic (∆Gt) and polyelectrolytic (∆Gpe) contribution. The polyelectrolytic contribution, ∆Gpe, may be determined using the relationship

∆Gpe ) -ZφRT ln([Na+])

(4)

The magnitude of ∆Gt will provide a measure of the contribution from the nonpolyelectrolytic forces to the free energy that stabilizes the dye-DNA complexes. These forces will include inter alia those from hydrogen bonding, van der Waals forces, hydrophobic transfer, etc. ∆Gpe is the polyelectrolytic contribution to the binding free energy arising from coupled polyelectrolytic effects, the most important being the release of the condensed counterions from the double helix due to the binding of the charged intercalator. Table 4 summarizes the comparative thermodynamic parameters of the binding of PSF and SO to poly(dG-dC) · poly(dG-dC) and poly(dG) · poly(dC). At the 20 mM [Na+] concentration employed in these studies, the values of ∆Gpe contribution to the free energy were all in the range -1.8 to -2.0 kcal mol-1. Under similar conditions, singly charged mono intercalators would typically have ∆Gpe contributions of -2 to -3 kcal mol-1.36 Thus, it can be seen that the nonpolyelectrolytic forces play a dominant role in the stabilization of the complexes of both PSF and SO with the two polynucleotides as ∆Gt was found to be about 73-75% of the total free energy (Supporting Information, Figure S4C,D). Temperature-Dependent Isothermal Calorimetric Data: Heat Capacity Changes. The constant pressure heat capacity change (∆Cp) of PSF and SO-polynucleotide interactions were determined by performing the titration at different temperatures and employing the standard relationship, ∆Cp ) δ∆H/δT, for poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), and poly(dAdT) · poly(dA-dT). Heat capacity change data can provide valuable insights into the type and magnitude of forces involved in the complexation. The thermodynamic parameters from ITC studies performed at three temperatures, viz., 10, 20, and 30 °C, are presented in Table 3. The association constant of the complexation decreased in each case as the temperature was raised. The free energy change, however, was minimal. On the other hand, there were remarkable changes in the enthalpy and entropy contributionsswhile the ∆H values increased, the T∆S values decreased (Table 3). The variation of ∆H with temperature is presented in Figure 9, A and B. The slopes of the lines gave values of -130, -92, and -110 cal/(mol K), respectively, for the binding of PSF and -101, -81, and -94 cal/(mol K),

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Saha et al. ring system and the binding should be energetically favorable.37e The relationship ∆Ghyd ) 80((10) × ∆Cp allows the calculation of the free energy contribution from the hydrophobic transfer step of binding of these molecules.35 Such values calculated for the binding of PSF and SO to poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), and poly(dA-dT) · poly(dA-dT) were found to be -10.4, -7.4, and -8.8, and -8.1, -6.5, and -7.5 kcal/ mol, respectively (Table 3). The magnitudes of these are clearly within the range that were observed for the binding of these molecules to natural DNA4b and that of many other nucleic acid intercalating molecules.8,37e-g The higher water of hydration associated with the AT sequences appears to be the cause of the higher observed ∆Ghyd for poly(dA-dT) · poly(dA-dT) compared to poly(dG) · poly(dC). Conclusions

Figure 9. Plot of variation of enthalpy of binding (∆H) with temperature for the binding of (A) PSF with poly(dG-dC) · poly(dGdC) ((crossed squares), poly(dG) · poly(dC) (b), and poly(dA-dT) · poly(dA-dT) (2), and (B) SO with poly(dG-dC) · poly(dG-dC) (X), poly(dG) · poly(dC) (O), and poly(dA-dT) · poly(dA-dT) (4) at 20 °C in 20 mM cacodylate buffer of pH 7.0.

respectively, for the binding of SO to poly(dG-dC) · poly(dGdC), poly(dG) · poly(dC), and poly(dA-dT) · poly(dA-dT) (Table 3). Negative heat capacity values have also been observed for these dyes binding to natural DNAs4b and for many small molecules binding to DNA and RNA.7,8,11,37 Further, a large ∆Cp value is usually associated with changes in hydrophobic or polar group hydration and is considered as an indicator of dominant hydrophobic interactions in the binding process. The heat capacity values are lower for SO with the three polynucleotides, reflecting that the two bulky methyl substituents in its structure disfavor the complexation in comparison to PSF. Change in solvent-accessible surface area has been shown to be a large component of ∆Cp from several studies.37e,38 Further, structured water like the water of hydrophobic hydration can be associated with large heat capacity and the release of such water associated with the transfer of the nonpolar groups into the interior of the duplex can contribute to a negative term to the ∆Cp. Many inferences can be drawn from these results. First, the values of ∆Cp are nonzero, indicating temperature dependence of the enthalpy change. Second, the values of ∆Cp in almost all cases fall within the range 100-500 cal/(mol K) or are very close to the lower limit that is frequently observed for many ligandnucleic acid interactions.37a,c,e It is known that AT base pairs have more water of hydration compared to GC base pairs. Third, the slightly higher ∆Cp values for these dyes binding to the poly(dA-dT) · poly(dA-dT) compared to poly(dG) · poly(dC) may suggest conformational differences in its structure and also differences in the disruption of the water structure around the base pairs of this sequence on complexation. Four types or modes of DNA recognition by small molecules, viz., sequencespecific, nonspecific, minimal sequence-specific, and structurespecific, have been proposed.37b Small negative ∆Cp values are considered to be associated with a minimal sequence-specific binding, and hence the slightly negative nonzero ∆Cp values that are observed for PSF and SO complexation with the three polynucleotides studied here appear to denote sequence-selective binding. For DNA intercalators, a large hydrophobic contribution to the binding free energy is expected due to their aromatic

The goal of this study was a detailed elucidation of the sequence selectivity and energetics of the binding of two important phenazinium dyes, phenosafranin and safranin O, from studies with synthetic GC and AT polynucleotides having defined sequence of base pairs. These two dyes differ in only two methyl substitutions and hence an interesting aspect was elucidating the role of bulky substituents on the chromophore on the structural aspect and energetics of binding. The results of the structural studies provide clear evidence for the strong binding with the polynucleotides obeying neighbor exclusion principle; the binding was stronger with the alternating and homopolynucleotides of GC sequences over the AT polynucleotides and for PSF over SO. Both these conclusions were further confirmed, unequivocally, by the competition dialysis assay. Energetics of the interaction from microcalorimetry revealed that the binding reaction was favored by both negative enthalpy and positive entropy changes, though to different extents. The entropy contribution was higher for SO in all the cases. The binding of PSF and SO to the alternating AT polynucleotide was favored by higher entropic contribution compared to that with the GC polynucleotides, indicating the stronger perturbation of the water structure associated with the AT base pairs. Homo AT polynucleotide showed anomalous thermodynamic behavior due to its unique structure. The binding enthalpy of the complexation became more favorable and binding entropy less favorable as the temperature increased. Negative heat capacity changes in all the three systems are correlated to the involvement of significant hydrophobic component in the complexation, corroborating the low polyelectrolytic contribution to the binding free energy. The differences in the ∆Cp values indicated important differences in the formation of the intercalative complexes by PSF and SO, suggesting the release of hydrophobic water of hydration. The thermodynamic results also implicate significant role for the steric bulk due to the methyl substitutions on the phenazinium moiety to account for the reduced binding of SO. The binding affinity was lower, the enthalpy of binding was lower, and the entropy contribution was higher compared to PSF. Furthermore, the heat capacity values were also lower, so also the ∆Ghyd values compared to PSF. This clearly proves that PSF being sterically smaller than SO can intercalate to a greater extent between the base pairs, a notion previously proposed from studies with natural DNAs.4b,5 Thus, this study presents an in-depth understanding of the sequence selectivity of these dyes, the energetics associated with the sequence selective interaction, and the role of bulkier substituents on the structural and energetic aspect of sequenceselective intercalation that further advances our knowledge on the interaction of small molecules in general to repeating

Phenazinium Dyes-Polynucleotide Complexation sequences of base pairs useful for designing better DNA binding molecules for therapeutic applications. Acknowledgment. This work was supported by grants from the network project on “Comparative genomics and biology of noncoding RNA in the human genome” (Project No. NWP 0036) of the Council of Scientific and Industrial Research (CSIR), Government of India. I.S. is a recipient of the Junior Research Fellowship of CSIR through the National Eligibility Test. M.H. is a Senior Research Fellow (NET) of the University Grants Commission, Government of India. The authors are grateful to Prof. Siddhartha Roy, Director, Indian Institute of Chemical Biology, for his patronage and all colleagues of Biophysical Chemistry Laboratory for their help and cooperation at every stage of this work. The critical comments of the two anonymous reviewers that enabled us to improve the manuscript are also highly appreciated. Supporting Information Available: Stern-Volmer plots for the quenching of PSF and SO and their complexes with the polynucleotides (Figure S1); plots of increase in helix contour length (L/L0) versus r for the complexation of poly(dGdC) · poly(dG-dC), poly(dG) · poly(dC), poly(dA-dT) · poly(dAdT), and poly(dA) · poly(dT) with PSF and SO (Figure S2); Job plots for the complexation of PSF and SO with the four polynucleotides (Figure S3); and van’t Hoff plots of log Kb versus log [Na+] and polyelectrolytic and nonpolyelectrolytic contribution of the binding free energy of PSF and SO to poly(dG-dC) · poly(dG-dC), poly(dG) · poly(dC), and poly(dAdT) · poly(dA-dT) (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Waring, M. J. Annu. ReV. Biochem. 1981, 50, 159–192. (b) Hurley, L. H. Biochem. Soc. Trans. 2001, 29, 692–696. (c) Hurley, L. H. Nature ReV. 2002, 2, 188–200. (d) Martines, R.; Chacon-Garcia, L. Curr. Med. Chem. 2005, 12, 127–151. (e) Palchaudhuri, R.; Hergenrother, P. J. Curr. Opin. Biotechnol. 2007, 18, 497–503. (f) Maiti, M.; Suresh Kumar, G. Med. Res. ReV. 2007, 27, 649–695. (g) Dufff, M. R., Jr.; Mudhivarthi, V. K.; Kumar, C. V. J. Phys. Chem. B 2009, 113, 1710–1721. (h) White, S.; Szewczyk, J. W.; Turner, J. M.; Baird, E. E.; Dervan, P. B. Nature 1998, 391, 468–471. (i) Dervan, P. B. Bioorg. Med. Chem. 2001, 9, 2215– 2235. (j) Reddy, B. S. P.; Sondhi, S. M.; Lown, J. W. Pharmacol. Ther. 1999, 84, 1–111. (2) (a) Broglia, M. F.; Gomez, M. L.; Bertolotti, S. G.; Montejano, H. A.; Previtali, C. M. J. Photochem. Photobiol. A 2005, 173, 115–120. (b) Gopidas, K. R.; Kamat, P. V. J. Phys. Chem. 1990, 94, 4723–4727. (c) Jokush, S.; Timpe, H. J.; Schnabel, W.; Turro, N. J. J. Phys. Chem. A 1997, 101, 440–445. (d) Jayanthi, S. S.; Ramamurthy, P. J. Chem. Soc., Faraday Trans 1998, 94, 1675–1680. (e) Niu, X. L.; Sun, W.; Jiao, K. Russ. J. Electrochem. 2009, 45, 967–971. (f) Priya, P. L.; Shanmughavel, P. Bioinformation 2009, 4, 123–126. (g) Sun, W.; You, J.; Hu, X.; Jia, K. Anal. Sci. 2006, 22, 691–696. (h) Gao, F.; Zhang, L.; Bian, G. R.; Wang, L. Spectrosc. Lett. 2006, 39, 73–84. (i) Huang, H. Y.; Wang, C. M. J. Phys. Chem C 2010, 114, 3560–3567. (j) Bose, D.; Ghosh, D.; Das, P.; Girigoswami, A.; Sarkar, D.; Chattopadhyay, N. Chem. Phys. Lipids 2010, 163, 94–101. (3) Balcarove, Z.; Kleinwatcher, Z.; Lober, G.; Luck, G.; Zimmer, C.; Klarner, R.; Smekal, E. Biophys. Chem. 1979, 9, 121–131. (4) (a) Das, S.; Suresh Kumar, G. J. Mol. Struct. 2008, 872, 56–63. (b) Saha, I.; Hossain, M.; Suresh Kumar, G. Phys. Chem. Chem. Phys. 2010, 12, 12771–12779. (5) Sarkar, D.; Das, P.; Basak, S.; Chattopadhyay, N. J. Phys. Chem. B 2008, 112, 9243–9249.

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Sequence-Selective Binding of Phenazinium Dyes Phenosafranin and

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