Circular dichroism of acridine orange bound to DNA

The circular dichroism (CD) spectra of DNA–acridine orange (DNA–AO) complex in the visible region were measured at DNA phosphate-to-dye ratios (P/D) from 1 to 550. The CD spectrum of DNA–AO complex in the P/D ratio between 1 and approximately 40 consists of four components, i.e., positive CD bands centered at 510 and 480 mμ, and negative CD bands at 497 and 468 mμ. The CD bands at 510 and 468 mμ are optimum at P/D = 4, and the change of ε₁ ? ε_r with P/D suggests that both of them are induced from the interaction between dye molecules bound to adjacent DNA binding sites, each of which is composed of four nucleotides. This is supported by the fact that the values of ε₁ ? ε_r for both decrease with increasing temperature or increasing methylene blue concentration added to the complex. The negative Cotton effect at, 497 mμ is most favored at larger P/D ratio (～8), and the suggested assignment is to the interaction between two dye molecules bound with an empty site between them. A positive Cotton effect at 480 mμ is observed at P/D ratio of less than 4 and is optimum at 1. Above P/D ratio of 40, the CD spectrum of the complex can not be resolved into its components and even at sufficiently high P/D ratio (550) the complex shows a small Cotton effect.     

Studies of the optical properties of the proflavine–DNA complex

We report studies of the optical properties of the proflavine–DNA complex, using absorbance and circular dichroism spectroscopy. From comparison of the absorption spectra of proflavine complexed with calf thymus and T2 DNA, we conclude that stacking of the dyes external to the double helix is comparatively much weaker with T2 DXA, probably because of its glucosylation. Several sources are found for the circular dichroism induced in proflavine when it is complexed with DNA. There is a relatively weak circular dichroism induced when the dye is infinitely dilute on the DNA lattice; this presumably arises from the environmental asymmetry of the binding site. Stronger circular dichroism effects are induced by interaction of intercalated and stacked dyes; studies with T2 DNA, for which stacking seems to be blocked, permit a tentative resolution of effects due to the two modes of binding. One recurring theme of these studies is the observation that the optical properties are quite dependent on environment. The most dramatic example is a strong variation with salt concentration of the amplitude of the circular dichroism induced in the isolated (intercalated) monomer by the surrounding DNA. This suggests that the structure of the intercalated complex is quite sensitive to external conditions.     

Calorimetry of DNA-dye interactions in aqueous solution: I. Proflavine and ethidium bromide

The results of an investigation on the interaction of proflavine and of ethidium bromide with DNA (calf thymus) in dilute aqueous solution are reported. The binding of the two dyes by DNA has been studied by means of microcalorimetric and of equilibrium dialysis measurements. Data on the thermodynamics of dimerization of both proflavine and ethidium bromide in aqueous solution obtained on the basis of spectroscopic and/or calorimetric experiments are also reported.The enthalpy data show that dye-dimerization and dye “strong” interaction with DNA are energetically favourable and quite similar while only in the latter case the phenomenon is also entropy driven. This is taken as further evidence in support of the concept that “strong” interaction-of both proflavine and ethidium bromide with DNA means dye molecules intercalation into the native, double helical structure of the biopolymer.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

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.     

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.     

The use of intercalating dye molecules in the study of chromatin structure

This paper reviews the field of chromatin structure studied by means of dye molecules believed to intercalate within DNA. The emphasis is on dyes whose binding properties are the best characterized (ethidium bromide (EB). actinomycin D(AMD), proflavine (PF)) but studies in which less common dye molecules are used are also considered. A comparison is made between the binding of these dyes to purified DNA and to whole or partially deproteinized chromatin in order to investigate both the availability of DNA in chromatin and the localization of chromosomal proteins or the DNA backbone.     

Structural transitions of chromatin at low salt concentrations: a flow linear dichroism study

We have studied the structure of nuclease-solubilized chromatin from Ehrlich ascites cells by flow linear dichroism (LD) using the anisotropic absorption of the DNA bases and of two intercalated dyes, ethidium bromide and methylene blue. It is confirmed that intercalation occurs preferentially in the linker part of the chromatin fiber, at binding ratios (dye/base) below 0.020. Using this information, we determined the orientation of the linker in relation to the average DNA organization in chromatin. The LD measurements indicate that the conformation of chromatin is considerably changed in the ionic strength interval 0.1-10 mM NaCl: with increasing salt concentration, the LD of the intrinsic DNA base absorption changes signs, from negative to positive, at approximately 2.5 mM NaCl. The LD of the intercalated dyes also changes signs, however, at a somewhat higher salt concentration. The results are analyzed in terms of possible allowed combinations of tilt angles of nucleosomes and pitch or tilt angles of linker DNA sections relative to the fiber axis, at different salt concentrations in the interval 0.1-10 mM NaCl. Two models for the salt-induced structural change of chromatin are discussed.     

Circular dichroism studies of ethidium bromide binding to the isolated nucleolus.

Circular dichroism in the 300-360 nm region and fluorescence induced by intercaltating binding of ethidum bromide to both DNA and RNA components were studied in isolated HeLa nucleoli. Both DNA and RNA compoents contribute to the induced dichroic elliticity. Digestion of nucleoli by RNase or DNase shows that most of the induced ellipticity comes from the DNA component. In nucleoli with an RNA/DNA = 0.8/1.0 the RNA component gives only 20% of the total ellipticity when measured at an ethidium bromide/DNA = 0.25. Spectro-fluorometric titration shows that ethidium bromide intercalates mostly into DNA in nucleoli. Both circular dichroism and fluorescence studies indicate that both DNA and RNA components in isolated nucleoli are less accessible to intercalating binding by ethidium bromide when compared to purified nucleolar DNA, DNA in chromatin or purified ribosomal RNA. Circular dichroic measurements of intercalating binding of ethidium bromide to to nucleoli may be used to study changes in nucleoli under different physiological or pathological conditions.     

Dielectric behavior of DNA--dye complex. II

The dielectric relaxation of native DNA and the effect of aminoacridine dyes, such as acridine orange (AO), proflavine (PF), and ethidium bromide (EB) have been investigated at different molar DNA phosphate (P) to dye (D) ratios in the frequency range 100 Hz–100 kHz. The static dielectric constant was observed to decrease with increasing binding of aminoacridines. This was interpreted as arising from the neutralization of the surface changes of the DNA molecules as a result of dye binding. At any P/D ratio the extent of charge neutralization was greatest for AO and least for the EB–DNA complex. The relaxation time (τ) for dye-bound DNA was greater compared to that for native DNA. This increase in τ was ascribed to the increase in the length of the dye-bound DNA. The maximum value of τ occurred at P/D = 20, 10, and 2 for AO-, PF-, and EB-treated DNA, respectively. The variation of τ at various levels of binding gave a qualitative idea about the conformational changes of DNA due to its binding with the dyes.     

Effect of organic solvents on the properties of the complexes of DNA with proflavine and similar compounds

In order to obtain information on the binding forces involved in the formation of the complex proflavine–DNA by the stronger process I, the stability of the complexes was investigated in the presence of various organic solvents, methanol, ethanol, n-propanol, isopropanol, formamide, dimethyl sulfoxide, p-dioxane, glycerol, and ethylene glycol. Quantitative data on binding in terms of K/n and r were obtained by means of absorption and fluorescence spectra, as well as by a thermal denaturation technique. All organic solvents used decrease the binding ability of the dye. The effectiveness of the solvents increases with their hydrocarbon content, but can hardly be related to their dielectric constant. The complex formation is effectively suppressed by organic solvent concentrations, in which DNA still preserves its double-helical conformation. These results demonstrate the importance of hydrophobic forces in the formation of the complex proflavine–DNA in aqueous solution. The similarity in spectroscopic properties of proflavine bound to DNA by process I and the same dye dissolved in an organic solvent make it possible to interpret the observed red shift of the long-wavelength absorption peak as being due to the interaction of the dye molecules with the less polar environment. The same behavior was found for other dyes capable of intercalation like purified trypaflavine, phenosafranine and ethidium bromide. However, intercalation is not a necessary condition, as it was shown in the case of pinacyanol, which binds only at the surface of DNA.     

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.     

Ethidium bromide-dinucleotide complexes. Evidence for intercalation and sequence preferences in binding to double-stranded nucleic acids.

The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine–purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.     

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.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.     

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.     

Effect of 5-iodo-2′-deoxyuridine on physical properties and nonhistone chromosomal proteins of chromatin from Burkitt somatic cell hybrids

A Burkitt somatic cell hybrid line, D98/HR-1, contains latent Epstein-Barr virus genome which can be induced to express by 5-iodo-2′-deoxyuridine (IdUrd). Chromatins were isolated from confluent monolayers of D98/HR-1 grown in the presence and absence of IdUrd and its effect on the chromatin was studied by circular dichroism, ethidium bromide binding capacity, and template activity. IdUrd-Treated chromatin consistently exhibits a 20 to 25% decrease in positive ellipticity of circular dichroism at 275 nm. Scatchard plots of spectrophotometric titration of chromatin with ethidium bromide indicate a 25% reduction of the number of primary binding sites in IdUrd-treated chromatin which corresponds with a 25% reduction in template activity of the same chromatin. These effects are reversible when IdUrd-treated cells were released into IdUrd-free medium for 3 h. However, the differences in circular dichroism spectra and dye binding capacity are not observed between DNA isolated from IdUrd-treated and nontreated cells. These results suggest that chromosomal proteins or modification of protein-DNA interactions are responsible for the alterations. Polyacrylamide gel electrophoretic analysis of the chromosomal proteins reveals that a class of nonhistone chromosomal protein in the high molecular weight region (above 130K) is lacking in IdUrd-treated cells.     

Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate.

Confluent monolayers of WI-38 diploid fibroblasts can be stimulated to proliferate by fresh serum. In the first 3 h after stimulation (that is, several hours before DNA replication) the chromatin of stimulated cells show structrual changes which include: (1) an increase in maximum positive ellipticity and a blue shift in the 250-300 nm region of circular dichroism spectra; and (2) an increase,in isolated chromatin, of the number of binding sites for the intercalating dye, ethidium bromide.The differences between chromtin of stimulated and chromatin of unstimulated cells are abolised when bother chromatins are treated with 0.25 M NaCL.     

Extrinsic Cotton effects of proflavine bound to polynucleotides

The magnitude of the Cotton effect of proflavine which is bound to RNA or to denatured DNA depends on the ratio of bound proflavine to nucleic acid base. A statistical treatment which explains this behavior has been fitted to the experimental curves and indicates that optical activity arises through interaction between two or more bound proflavine molecules. The corresponding requirement with double helical DNA is for interaction between 3–4 proflavine molecules. Although proflavine binds to denatured DNA at pH 2.8, as shown by the shift of the proflavine spectrum, the strong binding process is absent, and to this is attributed the absence of the Cotton effect at low pH. Studies on the Cotton effects of proflavine bound to poly A and poly U at neutral pH, to poly A at acid pH and to poly (A + U) allow the generalization that a relatively rigid configuration of the binding macromolecule is required for the induction of these extrinsic Cotton effects.