Induced circular dichroism of DNA-dye complexes

The binding of methylene blue, proflavine, and ethidium bromide with DNA has been studied by spectrophotometric titration. Methylene blue and proflavine or methylene blue and ethidium bromide were simultaneously titrated by DNA. The results indicate that all of these dyes compete for the same bindine sites. The binding properties are discussed in terms of symmetry. The optical properties of the dye–DNA complexes have been studied as a function of DNA/dye ratio. The induced circular dichriosm due to dye–dye interaction was measured at low dye/DNA ratios for cases involving both the same dye and different dyes. A positive Cotton effect for DNA–proflavine complex may be induced at 465 mμ by eithr proflavine or ethidium bromide, whereas a netgative Cotton effect at 465 mμ may be induced by methylene blue. The limiting circular dichroism, with no dye–dye interaction, and the induced circular dichroism spectra are discussed in terms of symmetry rules.     

Optical and electro-optical properties of the complexes of dibutylproflavine with DNA and nucleohistone

A number of optical and electro-optical measurements showed that the binding of dibutylproflavine to calf thymus DNA and nucleohistone in 1 mM NaCl could be resolved on the basis of two external and orientated bound species, whose binding parameters have been determined by a spectrophotometric method.The absorption and the electric dichroism properties were found to show up additivity of contributions from the two bound species. The orientation of the long axis of the strongly bound molecules of species I appeared close to the inclination of the DNA grooves, while that of species II differed by about ten degrees.A spatial proximity of the two binding sites was suggested by the dependence of the circular dichroism signals, the fluorescence quantum yield and the emission anisotropy on the degree of binding. The mobility of the first bound dibutylproflavine molecules was similar to that of the intercalated proflavine molecules, but lower in nucleohistone as compared to DNA.     

The circular dichroism of complexes of 2,7-di-tertiary-butyl proflavine with DNA

The binding isotherm of 2, 7-di-tert-butyl proflavine on calf thymus DNA has been measured by dialysis equilibrium. The CD spectra of complexes of the dye and DNA have been measured, and the variation of the induced circular dichroism of the dye with the amount of dye bound (r) has been found. The results show that di-tert-butyl proflavine binds to DNA in a completely different manner from proflavine itself, since both the visible and ultraviolet CD spectra of complexes of the two dyes with DNA differ markedly. The conformation of the nucleic acid is not affected by the binding of di-tert-butyl proflavine. It is possible that these results may allow determination, by using CD spectroscopy, of whether molecules intercalate into DNA.     

Study on the interaction between 4-(2-diethylamino-ethylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile and DNA by molecular spectra

The binding geometry of a heterocyclic compound, 4-(2-diethylamino-ethylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (A1) to CT DNA was studied by molecular spectroscopy. Deduced from SYBR Green-DNA melt curve, UV-vis spectroscopy, and fluorescence studies, there were two different interaction mechanisms involved in the whole interaction process depending on the R-value (R, the molar ratio of A1 to CT DNA base pairs). The value R = 0.20 was the turning point. The induced circular dichroism (ICD) spectra of A1 complexed with CT DNA, poly[(G-C)2] and poly[(A-T)2] showed when R < or = 0.20, A1 intercalated into CT DNA and the intercalation orientation of A1 to the dyad axis of DNA double-helix was heterogeneous. When R > 0.20, stacking of A1 on surface helix of DNA occurred driven by the protonation of amidogen group in the N,N-diethyldiamine substitution of A1, which was illustrated by the changes of A1-DNA geometry in different pH solutions. The intrinsic circular dichroism (CD) spectra showed the conformation of DNA converted from the B-form to A-like conformation due to the A1 intercalation.     

Cationic 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin fully intercalates at 5'-CG-3' steps of duplex DNA in solution

The interaction of 5,10,15, 20-tetrakis(N-methylpyridinium-4-yl)porphyrin (T4MPyP(4+)) with the oligonucleotide DNA duplex [d(GCACGTGC)](2) was studied by two-dimensional (1)H NMR spectroscopy, optical absorbance, circular dichroism, and molecular dynamics simulation employing particle mesh Ewald methods. T4MPyP(4+) is one of the largest aromatic molecules for which intercalative binding to DNA has been proposed, although this has been called into question by recent X-ray crystallographic work [Lipscomb et al. (1996) Biochemistry 35, 2818-2823]. T4MPyP(4+) binding to [d(GCACGTGC)](2) produced a single set of (mostly) upfield-shifted DNA resonances in slow exchange with the resonances of the free DNA. Intra- and intermolecular NOEs observed in the complex showed that the porphyrin intercalates at the central 5'-CG-3' step of the DNA duplex without disrupting the flanking base pairs. Absorption and circular dichroism spectra of the complex also support intercalative binding. Molecular dynamics simulations (using explicit solvent and PME methods), carried out for fully and partially intercalated complexes, yielded stable trajectories and plausible structures, but only the symmetrical, fully intercalated model agreed with NOESY data. Stable hydrogen bonding was observed during 600 ps of MD simulation for the base pairs flanking the binding site.     

Copper insertion facilitates water-soluble porphyrin binding to rA.rU and rA.dT base pairs in duplex RNA and RNA.DNA hybrids

The binding of the copper(II) complex of water-soluble meso-tetrakis(N-methylpyridinium-4-yl)porphyrin (TMPyP) to double-helical polynucleotides has been studied by optical absorption, circular dichroism (CD), and resonance Raman spectroscopic methods. The target polymers were RNA and RNA.DNA hybrids consisting of rA.rU, rI.rC, rA.dT, and rI.dC base pairs. Relative to the metal-free H(2)TMPyP [Uno, T., Hamasaki, K., Tanigawa, M., and Shimabayashi, S. (1997) Inorg. Chem. 36, 1676-1683], CuTMPyP binds to poly(rA).poly(dT) and poly(rA).poly(rU) with a greatly increased binding constant. The external self-stacking of the porphyrin on the surface of the polymers was evident from the strong conservative-type induced CD signals. The signal intensity correlated almost linearly with the number of stacking sites on the polymer except for poly(rA).poly(dT), which showed extraordinarily strong CD signals. Thus, the bound porphyrin may impose an ordered architecture on the polymer surface, the stacking being facilitated by the more planar nature of the CuTMPyP than the nonmetal counterpart. Resonance Raman spectra of the stacked CuTMPyP were indistinguishable from those of the intercalated one with positive delta(Cbeta-H) and negative delta(Cm-Py) bending shifts, and hence the stacked porphyrins are suggested to adopt a similar structure to that of intercalated ones. Porphyrin flattening by copper insertion opens a new avenue for medical applications of porphyrins, blocking biological events related to RNA and hybrids in malignant cells.     

Study of the interaction of DNA and acridine orange by various optical methods

The degree of binding of acridine orange to DNA, native or denatured, has been determined by equilibrium dialysis in 0.1M and 0.001M NaCl at 20°. The nature of the binding process has been investigated by studying various optical properties of the dye–DNA complexes and by relating them to the binding ratio. All these properties were found to vary quantitatively and qualitatively according to the successive stages of the process. These stages were assumed to be a strong binding of intercalated monomers followed by formation of bound dimers and finally by external binding of aggregates of native DNA. Absorption spectra of the complexes could be interpreted on that basis. Circular dichroism spectra were resolved into components: one band for intercalated monomers without interactions, two excition splittings for interacting monomers and bound dimers, respectively, weak bands and exciton splitting for external aggregates. The fluorescence intensity was greatly enhanced in intercallated monomers; its quenching at higher binding ratio was quantitatively related to dimer fixation. The value of the anisotropy of fluorescence at low binding ratio suggested a limited mobility of intercalated monomers; the decrease of polarization at higher binding was attributed to energy transfer between monomers. Electric dichroism displayed by the complexes in the dye absorption bands indicated an orientation of the bound molecules quite parallel to the base rings at low binding. In the range of fixation of dimers and external molecules, the dichroism was lower but still indicated an important degree of ordering.     

Thermal denaturation of the DNA-ethidium complex. Redistribution of the intercalated dye during melting.

The temperature dependence of the circular dichroism of the DNA-ethidium bromide complex at elevated temperatures provides evidence that the optical activity of the complex near 307 nm originates from interactions between intercalated dye molecules while the optical activity near 515 nm results from singly intercalated ethidium bromide molecules. The behavior of the circular dichroism of the complex at elevated temperatures also explains the higher ellipticities near 307 nm which characterize complexes formed between ethidium bromide and denaturated DNA. Finally the circular dichroism data indicate that the melting of the complex takes place in a stepwise manner with some DNA regions, probably AT-rich regions, dissociating first. The implications of these findings regarding the inhibiting effect of ethidium bromide on the function of DNA polymerase are examined.     

Interaction of ethidium bromide with DNA. Optical and electrooptical study

The binding of ethidium bromide to DNA has been studied by various optical methods. From fluorescence polarization studies, and film, electric linear dichroism, and circular dichroism spectra, we propose assignments of the absorption bands of the dye, which are discussed in connection with wave-mechanical calculations recently reported. The optical activity induced in the dye absorption bands upon binding to DNA was attributed to various origins depending on the electronic transition considered. The visible absorption band displayed a circular dichroism due to the asymmetry of the binding site and independent of the amount of binding. The transition identified at 378 nm from the circular dichroism and electric dichroism observations was thought to be due to a magnetic-dipole transition. It remained constant with increasing amounts of dye bound. The main ultraviolet band showed circular dichroism characteristics corresponding to exciton interactions between dye molecules bound to neighboring sites. The electric dichroism observed for the strongly bound dye molecules indicated that the phenanthridinium ring of ethidium bromide was probably not perfectly parallel to the DNA base planes. When the amount of dye bound to DNA exceeded the maximum amount compatible with the exclusion of adjacent binding sites, the electric dichroism decreased owing to the appearance of externally bound dye molecules with no contribution to the dichroism. Sonicated DNA was used to study the lengthening of the DNA molecule upon complexation. Although the viscosity of the complexes increased with the amount of binding, the rotational diffusion coefficient measured by the electric birefringence relaxation was not detectably affected. The absence of variation in the electric birefringence with the binding indicated that the DNA base stacking remained unaltered.     

Relaxation kinetics of the interaction between RNA and metal-intercalators: the Poly(A).Poly(U)/platinum-proflavine system

The interactions of Poly(A).Poly(U) with the cis-platinum derivative of proflavine [{PtCl(tmen)}(2){HNC(13)H(7)(NHCH(2)CH(2))(2)}](+) (PRPt) and proflavine (PR) are investigated by spectrophotometry, spectrofluorimetry and T-jump relaxation at I=0.2M, pH 7.0, and T=25 degrees C. Base-dye interactions prevail at high RNA/dye ratio and binding isotherms analysis reveals that both dyes bind to Poly(A).Poly(U) according to the excluded site model (n=2). Only one relaxation effect is observed for the Poly(A).Poly(U)/PRPt system, whereas two effects are observed with Poly(A).Poly(U)/PR. The results agree with the sequence D+S <==> D, S <==> DS(I) <==> DS(II), where D,S is an external complex, DS(I) is a partially intercalated species, and DS(II) is the fully intercalated complex. Formation of DS(II) could be observed in the case of proflavine only. This result is interpreted by assuming that the platinum-containing residue of PRPt hinders the full intercalation of the acridine residue.     

Study of the binding of single-stranded DNA-binding protein to DNA and poly(rA) using electric field induced birefringence and circular dichroism spectroscopy

Binding of the single-stranded DNA-binding protein (SSB) of Escherichia coli to single-stranded (ss) polynucleotides produces characteristic changes in the absorbance (OD) and circular dichroism (CD) spectra of the polynucleotides. By use of these techniques, complexes of SSB protein and poly(rA) were shown to display two of the binding modes reported by Lohman and Overman [Lohman, T.M., & Overman, L. (1985) J. Biol. Chem. 260, 3594-3603]. The circular dichroism spectra of the "low salt" (10 mM NaCl) and "high salt" (greater than 50 mM NaCl) binding mode are similar in shape, but not in intensity. SSB binding to poly(rA) yields a complexed CD spectrum that shares several characteristics with the spectra obtained for the binding of AdDBP, GP32, and gene V protein to poly(rA). We therefore propose that the local structure of the SSB-poly(rA) complex is comparable to the structures proposed for the complexes of these three-stranded DNA-binding proteins with DNA (and RNA) and independent of the SSB-binding mode. Electric field induced birefringence experiments were used to show that the projected base-base distance of the complex is about 0.23 nm, in agreement with electron microscopy results. Nevertheless, the local distance between the successive bases in the complex will be quite large, due to the coiling of the DNA around the SSB tetramer, thus partly explaining the observed CD changes induced upon complexation with single-stranded DNA and RNA.     

The binding of 9-aminoacridine to calf thymus DNA in aqueous solution. Electronic spectral studies

Absorption, circular dichroism and steady-state fluorescence spectra were determined of 9-aminoacridine solutions in the presence of DNA at an ionic strength of 0.001 mol dm-3. Up to a dye/DNA phosphorus ratio of about 0.2 the results are fully consistent with the requirements and predictions of a binding model already shown to apply to the binding of other aminoacridines to DNA. The apparently anomalous spectroscopic behaviour of the 9-aminoacridine/DNA system compared with proflavine/DNA, for example, can be satisfactorily explained from a consideration of the magnitudes of exciton interactions between dyes bound to DNA.     

On the complex formation of acridine dyes with DNA. VII. Dependence of the binding on the dye structure

The binding constants for the complex formation of more than twenty ring nitrogen-and amino-substituted acridine derivatives with calf thymas DNA were measured by a fluorescence method. DNA quenches the fluorescence of the aminoacridine dyes so long as both amino hydrogens are not substituted. These dyes show an enhancement of their fluorescence intensity in the presence of DNA. Typical representatives of both are proflavine and acridine orange derivatives, respectively. A discussion of steric and electronic influences of various substituents attached to the ring nitrogen and amino groups on the binding led to the concept of different conformations for intercalated acridines without amino groups and the aminoacridines. The electrostatic binding site of the former seems to be the positively charged ring nitrogen, while the binding sites in the aminoacridines are so located that the amino groups are directed towards the negatively charged DNA phosphates.     

A clarification of the complex spectrum observed with the ultraviolet circular dichroism of ethidium bromide bound to DNA.

Ethidium bromide intercalation strongly effects the circular dichroism spectrum of DNA in the region of 230-300 mu, in a complex manner. In this report we present a study that quantitizes the relationships of the circular dichroism spectrum in the region of 230-300 mu and the ethidium bromide induced optical activity centered around 308 mu. We present evidence of two hidden cooperative bands that are probably the negative counterparts of the 308 mu band and 330 mu shoulder positive cooperative bands. The hidden band is quantitatively characterized. We confirm that the direct effect of ethidium bromide on the DNA spectrum is simply linearly proportional to the amount of intercalated dye. We also observe that the ethidium bromide enters freely when there is a molecule intercalated for every 3 sites, but that the intercalation is more difficult when the molecule intercalates at every second site.     

The induced circular dichroism of proflavine intercalated to DNA. Dye-polymer exciton interactions

The circular dichroism induced in the visible absorption band of proflavine cation isolatedly intercalated to DNA was investigated in terms of the dye-DNA base pair exciton interaction. The remarkable ionic strength dependence of the induced CD magnitude was in good accord with the CD magnitude calculated on the basis of the dye-polymer Frenkel exciton interaction model and under the extent of helix deformation required for intercalation. In particular the application of the internal and modified intercalation models coupled with the deep trap approximation implied that the preference of the modified intercalation due to electrostatic interaction between the acridine-nitrogen atom and the DNA phosphate group is combined with relatively high ionic strength compared with the internal intercalation.     

Binding mode of porphyrins to poly[d(A-T)(2)] and poly[d(G-C)(2)

We examined the binding geometry of Co-meso-tetrakis (N-methyl pyridinium-4-yl)porphyrin, Co-meso-tetrakis (N-n-butyl pyridinium-4-yl)porphyrin and their metal-free ligands to poly[d(A-T)(2)] and poly[d(G-C)(2)] by optical spectroscopic methods including absorption, circular and linear dichroism spectroscopy, and fluorescence energy transfer technique. Signs of an induced CD spectrum in the Soret band depend only on the nature of the DNA sequence; all porphyrins exhibit negative CD when bound to poly[d(G-C)(2)] and positive when bound to poly[d(A-T)(2)]. Close analysis of the linear dichroism result reveals that all porphyrins exhibit outside binding when complexed with poly[d(A-T)(2)], regardless of the existence of a central metal and side chain. However, in the case of poly[d(G-C)(2)], we observed intercalative binding mode for two nonmetalloporphyrins and an outside binding mode for metalloporphyrins. The nature of the outside binding modes of the porphyrins, when complexed with poly[d(A-T)(2)] and poly[d(G-C)(2)], are quite different. We also demonstrate that an energy transfer from the excited nucleo-bases to porphyrins can occur for metalloporphyrins.     

Characterization of the binding of YO to [poly(dA-dT)]2 and [poly(dG-dC)]2, and of the fluorescent properties of YO and YOYO complexed with the polynucleotides and double-stranded DNA

The interaction between the fluorescent dye YO (oxazole yellow) and the alternating polynucleotides [poly(dA-dT)]₂[the duplex of alternating poly(dA-dT)]and [poly(dG-dC)]₂[the duplex of alternating poly(dG-dC)] has been studied with optical spectroscopic techniques including absorbance, flow linear dichroism, CD, and fluorescence measurements. The principal features of the spectra are very similar for the two polynucleotide solutions, showing that YO binds quite similarly to AT and GC base pairs. From a strongly negative reduced linear dichroism (LD^r) in the dye absorption band, an induced negative CD, and transfer of energy from the bases to bound YO, we conclude that at low mixing ratios YO is intercalated in both [poly(dA-dT)]₂ and [poly(dG-dC)]₂. At higher mixing ratios an external binding mode starts to contribute, evidenced from the appearance of an exciton CD. The conclusion that YO binds in a similar way to AT and GC base pairs should be valid also for the dimer YOYO since its YO units have been found to bind to double-stranded (dsDNA) in the same way as the YO monomer. The fluorescence properties of YO and YOYO complexed with DNA or the polynucleotides have been characterized by studying the dependence of fluorescence intensity on temperature, mixing ratio, and ionic strength. The fluorescence intensity and fluorescence lifetime of YO-DNA decrease strongly with increasing mixing ratio, whereas the fluorescence intensity of YOYO-DNA shows a weaker dependence, indicating that the quantum yield depends on the distance between the YO chromophores on the DNA chain. Further, the fluorescence intensity of YO depends on the base sequence; the quantum yield and fluorescence lifetime for YO complexed with [poly(dG-dC)]₂ are about twice as large as for YO complexed with [poly(dA-dT)]₂. Measurements of excitation spectra at different mixing ratios and different emission wavelengths indicate that the fluorescence of the externally bound chromophores is negligible compared to the intercalated ones. © 1995 John Wiley & Sons, Inc.     

Linear dichroism studies of binding site structures in solution: Complexes between DNA and basic arylmethane dyes

The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K₁ ca 10⁵ M^?1; site density per nucleotide base n₁ ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50° to the local DNA helix axis. The complex with fuchsin is weaker (10⁴M^?1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C_2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation.     

Studies on the structure of chemically methylated DNA

CD and melting temperature measurements on the nature of DNA with chemically methylated guanine-rich sites indicate that the stable secondary structure of DNA depicted by Ramstein et al- involves considerable distortions resulting from decreased base-base stacking interaction. Besides that quantum chemical data gained from PPP calculations are in favor of a weaker hydrogen bonding interaction in the methylated guanine-cytosine base pair. CD measurements demonstrate that methylated DNA-regions differ from the nonmethylated helical structure, since formation of a condensed conformation as occurs in the transition from B to the C-uke structure is prevented by positively charged methylated guanine residues. An increase in helix winding angle, however, can not be excluded. Binding ability of the dyes acridine orange, phenosafranine, and the antibiotic actinomycin C is lowered for methylated DNA, while binding of proflavine is, in accordance with the results of Ramstein and Leng, slightly enhanced. The reason for the opposite behavior of proflavine is at present not fully understood. In particular changes in the binding ability with dyes could not be correlated with base specificity of complex formation. It is discussed that structural changes in DNA towards a loose conformation decrease the binding tendency for acridine orange, phenosafranine, and actinomycin C.     

DNA binding, DNA cleavage, and cytotoxicity studies of two new copper (II) complexes

The DNA binding behavior of [Cu(phen)(phen-dione)Cl]Cl (1) and [Cu(bpy)(phen-dione)Cl]Cl (2) was studied with a series of techniques including UV-vis absorption, circular dichroism spectroscopy, and viscometric methods. Cytotoxicity effect and DNA unwinding properties were also investigated. The results indicate that the Cu(II) complexes interact with calf-thymus DNA by both partially intercalative and hydrogen binding. These findings have been further substantiated by the determination of intrinsic binding constants spectrophotometrically, 12.5?×?10(5) and 5?×?10(5) for 1 and 2, respectively. Our findings suggest that the type of ligands and structure of complexes have marked effect on the binding affinity of complexes involving CT-DNA. Circular dichroism results show that complex 1 causes considerable increase in base stacking of DNA, whereas 2 decreases the base stacking, which is related to more extended aromatic area of 1,10-phenanthroline in 1 rather than bipyridine in 2. Slow decrease in DNA viscosity indicates partially intercalative binding in addition to hydrogen binding on the surface of DNA. The second binding mode was also confirmed by additional tests: interaction in denaturation condition and acidic pH. Also, these new complexes induced cleavage in pUC18 plasmid DNA as indicated in gel electrophoresis and showed excellent antitumor activity against K562 (human chronic myeloid leukemia) cells.