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.     

Effect of chemical structures of acridine and triphenylmethane dyes on the induced optical activity of DNA-dye complexes

Fifteen symmetrically substituted acridine dyes, all of which are interrelated by their chemical structures, each belonging to a C_2v symmetry, and three triphenylmethane dyes with amino or dimethylamino substituents are utilized to study necessary conditions for the appearance of extrinsic Cotton effects upon their binding to native and heat-denatured deoxyribonucleic acid (DNA). Three different kinds of the DNA–dye complexes, i.e., (1) dye added to native DNA, (2) heat-denatured DNA–dye complex, and (3) dye added to preheated DNA, were examined for each dye at a fixed P/D value of about 4. Optical activity was always observed for the compelexes of type (1) in each absorption band of the dyes in the visible and near-ultraviolet region. Two exceptions are 9-acetamido- and 9-hydroxyacridine, both being nonionic in aqueous solution at a pH range of 6. Acridinium chloride was unable to exhibit any definite extrinsic Cotton effect for complexes (2) and (3). Thus, the monocationic form of a dye due to the protonation or quaternization of the ring nitrogen in acridines or exonuclear amino nitrogen in triphenylmethane dyes is concluded to be an essential factor for extrinsic Cotton effect to appear. Changes in the absorption spectra upon complex formation are also related to the structure of dyes. Hypochromism and bathochromism are associated with the induced optical activity in all cases in the presence of native and denatured DNA.     

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.     

Ultraviolet spectra of the complexes of acridine orange with deoxyribonucleic acid and polyphosphates

Summary Two kinds of changes were found in ultraviolet spectrum of acridine orange bound to polyphosphate and native or denatured DNA: (a) changes similar to those caused by aggregation in the solutions of pure acridine orange (i.e. blue shifts of the bands at 37300 cm^–1 and 43670 cm^–1, a decrease of absorbance of the band at 34600 cm^–1 and an increase of absorbance of the band at 43670 cm^–1), which were observed at those ratiosP/D, when the dye formed aggregates on the surface of the polyanion; (b) a decrease of absorbance in the whole near ultraviolet region, which had high value even when isolated dye molecules were bound to the polyanion. While the first kind of changes is due to mutual interactions between the aggregated acridine orange molecules, the second kind can be explained as due to interaction of the dye molecules with adjacent chromophores of the polyanion and/or solvent. The maximum value of the hypochromic effect in the near ultraviolet maximum was higher for complexes of denatured DNA than for complexes of native DNA.     

Proton and phosphorus nuclear magnetic resonance studies of an oligothymidylate covalently linked to an acridine derivative and of its binding to complementary sequences

An oligodeoxynucleotide containing four thymines and covalently attached to an acridine derivative through its 3'-phosphate [(Tp)4(CH2)5Acr] was synthesized. Its conformation in solution was investigated by proton magnetic resonance. Both intramolecular interactions between the acridine dye and thymines and intermolecular interactions were demonstrated. Both proton and phosphorus magnetic resonances were used to study the specific interaction of (Tp)4(CH2)5Acr with poly(rA) and (Ap)3A. The results were compared to those obtained when the acridine-containing substituent was replaced by an ethyl group attached to the 3'-phosphate of the oligothymidylate. The acridine dye strongly stabilized the complexes formed with both poly(rA) and (Ap)3A. Upfield shifts of both adenine and acridine proton resonances were observed in the complexes. These results were ascribed to an intercalation of the acridine ring between A X T base pairs of the duplex structure formed by the oligothymidylate with its complementary oligoadenylate sequence. An analysis of proton and phosphorus chemical shifts as well as measurements of T1 relaxation times at different temperatures allowed us to propose several structures for the complexes formed by (Tp)4(CH2)5Acr with its complementary sequence.     

Effect of gamma-irradiation on dye-DNA binding.

The effect of gamma-rays on the binding of proflavine and acridine orange to DNA was investigated by spectrophotometry. The effect of irradiation was observed on the buffered solutions of the free dye and free DNA. A dose of about 35 krad caused a hyperchromicity of 30-40 per cent to the DNA peak at 258 nm, while the same dose introduced a hypochromic effect to the monomer peaks of the dyes by 30 per cent. This implied that gamma-rays have an effect of decreasing the monomer concentration of free-day molecules in solution. From the results, we conclude that more dye is bound to the changed conformation of dye-bound DNA on irradiation. Scratchard-binding isotherms drawn for the unirradiated and irradiated complexes of Pf-DNA showed interesting differences. Similar isotherms could not be obtained for the acridine orange-DNA system.     

Induced Cotton effects of tRNA-acridine orange complex and tRNA conformation

Circular dichroism spectra of acridine orange bound to E. coli tRNA were studied at varying tRNA phosphate-to-dye (P/D) ratios for both unfractionated and purified materials in the absence of Mg⁺⁺. From the rather discrete features exhibited in the circular dichroism spectra three types of interactions were observed: (1) A high P/D ratio such as 75.2 or 49.8 indicates the interaction between the nucleotide base and dye molecule. The spectra with a large positive peak at 515 mμ are, however, quite different from that of DNA–AO complex under similar conditions. (2) With an intermediate P/D ratio (26.5 to 9.6) dye molecules bound strongly to the polynucleotide chain. (3) With low P/D ratios (≤7.5) the interaction appears to be due to the stacked dye molecules in the single-stranded part of tRNA. The spectra of the third group have an isobestic point at 477 mμ. Below a P/D ratio of 4 the spectrum shows one positive and two negative bands which may be the characteristics of circular dichroism of stacked dyes in polynucleotide chain. Although no drastic change in the conformation of tRNA itself was detectable in the presence of Mg⁺⁺ in the ultraviolet region, a dramatic change was observed in the circular dichroism of tRNA–acridine orange complex when Mg⁺⁺ concentration was increased to 10^?3M. It was inferred that certain conformational changes other than simple hydrogen bond formation occured in tRNA molecules at this high Mg⁺⁺ concentration, so that the amount of bound dye in the stacking condition was increased through the transition.     

Identification of the 9-aminoacridine/DNA complex responsible for photodynamic inactivation of P22

Acridine dyes bound to the condensed DNA within phage particles sensitize them to inactivation by visible light. The mechanism involves absorption of photons by an acridine/DNA complex, generating singlet oxygen, which covalently damages nearby proteins needed for DNA injection [Bryant, J., & King, J. (1985) J. Mol. Biol. 180, 837-863]. Acridines and related dyes interact with double-stranded DNA through a number of binding modes. To determine in condensed phage DNA the binding mode responsible for this inactivation, we have studied the formation of the DNA/acridine target complexes for photoinactivation. Analysis of the kinetics of 9-aminoacridine binding to Salmonella phage P22 particles revealed the formation of two binding species, one of which appeared more rapidly and was apparently an intermediate in the formation of the second. The rapidly forming species represented DNA sites with intercalated acridines, while the more slowly forming species represented the subsequent binding of additional acridine molecules to the DNA backbone of sites already containing intercalated dye. The rates of photoinactivation correlated with the rate of binding of 9-aminoacridine to the DNA backbone. This suggests that the most effective species for sensitizing phage to light-induced damage has acridine molecules stacked alongside the backbone of a region with intercalated molecules.     

Induced optical activity of acridine orange bound to poly-S-carboxymethyl-L-cysteine

The absorption and rotatory properties of acridine orange-poly-S-carboxymethyl-L -cysteine system in water and in 0.2 M NaCl have been measured at different pH and polymer-to-dye mixing ratios. The absorption spectra indicate that the dyes are bound to the polymer in dimeric or highly aggregated forms. At neutral pH where the polymer is randomly coiled, no optical activity is induced on the absorption bands of bound acridine orange. At acid pH where the polymer has the β-conformation, a pair of positive and negative circular dichroic bands occur at each of the absorption bands, centered around 458 and 261 mμ. The signs of those bands are opposite to those found for α-helical poly-L -glutamic acid. A model for the binding of dye to the β-form polymer is presented, in which dimeric dyes are attached to ionized carboxyl groups and slack one another to form linear arrays on both sides of an extended polypeptide chain. The observed circular dichroism spectra can be explained by the Tinoco's exciton mechanism, based on this model. Low molecular weight poly-S-carboxymethyl-L -cysteine induces quite a different circular dichroism on bound acridine orange.     

Structure and stability of complexes formed by nucleic acids. I. Binding of acridine to DNA

Nuclear magnetic resonance spectra of acridine have been measured in aqueous methanol solutions over a wide concentration range in the presence and absence of dissolved DNA. In solutions containing DNA the acridine spectra show a marked line broadening and intensity decrease at temperatures lower than 50°C. These line-shape changes can be associated with two types of binding interactions: (1) a tight, irrotational binding of the acridine at low acridine:phosphate ratios and (2) a weaker, rotationally less restrictive binding at high acridine concentrations. At temperatures above 50°C. a marked line narrowing is noted for the acridine spectrum and is attributed to an increase in mobility of the bound acridine as the DNA complex undergoes a helix–coil transition. A loose association of acridine molecules with the purine and pyrimidine bases in heat-denatured DNA is indicated by chemical shift changes in the acridine spectrum. The NMR measurements also show that the presence of acridine in denatured DNA solutions greatly reduces renaturation of the DNA.     

Structure and optical activity of the DNA-aminoacridine complex

The electronic absorption and circular dichroism spectra of the DNA-acridine orange complex have been measured over a range of ionic strength, pH, and DNA phosphate to dye (P/D) ratios. Three circular dichroism bands associated with the long wavelength absorption band of acridine orange are induced on complex formation with DNA. Two of the dichroism bands, due mainly to dimeric dye molecules, are favored by low ionic strength, low pH (3.2), and a low P/D ratio (～3), while the third, deriving primarily from monomeric dye, is optimum at high ionic strength, neutral pH, and a larger P/D ratio (9). The data suggest that monomeric acridine orange binds to DNA in the form of a left-handed helical array with four dye molecules per turn, while the bound dimer has a skewed sandwich conformation which is itself dissymmetric. The stereochemical relations between the bound monomer dye and the DNA are consistent with a modified intercalation model for the DNA-acridine complex.     

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.     

The circular dichroism in the ultraviolet of aminoacridines and ethidium bromide bound to DNA

The circular dicrosim (CD) spectra of complexes of DNA with ethidiun bromnide, profiavine, 9-aminoacridine and 4-etliyl-9-amino-acridine have been determined between 220 and 450 nm, the range lieing extended to 600 nm for ethidiufm bromide. The variation of the magnitude of the visible and near—ultraviolet CD spectra of ethidium bromide—DNA complexes with the amount of ligand bound (r) suggests a common binding position with profiavine. On the other hand, 4-ethyl-9-aminoacndine complexed to DNA shows CD spectra not distinguishable from those of 9-aminnoacnidmc in both the visible and ultraviolet. The interpretation of these results with respect to the stereochemistry of the DNA-ligand complexes is discussed.     

Interaction of polynuclear aromatic hydrocarbons, 4-nitroquinoline 1-oxides, and various dyes with DNA

By using the flow dichroism method, in vitro interaction of the aromatic polynuclear hydrocarbons with calf thymus DNA was studied. A clear dichroism of the hydrocarbons which interacted with DNA was observed, showing a specific type of interaction. Hydrocarbons were classified into two groups, depending on whether the orientation is parallel or perpendicular to the direction of flow. 3,4-Benzopyrene, pyrene, and phenanthrene belong to the first group, and the second group contains 20-methylcholanthrene, tetracene, pentacene, and coronene. 4-Nitroquinoline 1-oxide (4-NQO) and its derivatives have also been found to be oriented parallel to the planes of bases of DNA, and a distinct parallel was found between the amount of binding and carcinogenicity. Base specificity of binding of 4-NQO with DNA-was studied, and purines were suggested to be active sites of interaction. Actinomycin D, several acridine dyes, and methylene blue etc. were found to be oriented parallel to the planes of DNA bases, and in the cases of acridine dyes this finding does not contradict the intercalation mechanism proposed by Lerman.     

Induced circular dichroism of acridine orange bound to double-stranded RNA and transfer RNA

The induced circular dichroism (CD) in the visible region of acridine orange bound to the double-stranded RNA from cytoplasmic polyhedrosis virus and to yeast tRNA has been measured as a function of RNA phosphate-to-dye ratio (P/D), under the conditions of 0.01 M Na⁺ at pH 7.0. The shape of the CD spectrum of acridine orange bound to the double-stranded RNA was quite different from the spectrum of the dye bound to DNA. The CD spectral features of acridine orange bound to the double-stranded regions in tRNA closely resembled those of the double-stranded RNA-dye complex, suggesting that the dyes bind similarly to the two RNA's. It was further found that the CD spectrum of the tRNA-dye complex at sufficiently high P/D ratios, which is assignable to monomeric, intercalated dye to the base-paired parts in tRNA, is also distinct from the corresponding spectrum of the DNA-dye complex.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Interaction of aminoacridines with deoxyribonucleic acid: viscosity of the complexes

The intrinsic, viscosities at zero shear rate of defined complexes of proflavine, 9-aminoacridine, and 9-amino-l,2,3,4-tetrahydroaeridine with calf thymus DNA have been determined at, various ionic strengths by means of rotating cylinder viscometers. By controlled adjustment, of the composition of the mixtures, the amount of bound acridine (r moles/g.-atom DNA phosphorus) was maintained constant at different dilutions. The intrinsic viscosities of the complexes increased with r up to r values (ca. 0.16–0.20) corresponding to the end of the process of strong binding of the acridinium cations. However, complex formation between the acridines and thermally denatured DNA caused either a marked decrease in viscosity (at the low ionic strengths of 0.0015 and 0.005) or no change at all (ionic strength 0.1). These results are discussed in the light of presently available hydrodynamic theories relating the intrinsic, viscosity of DNA to its molecular extension.     

Circular dichroism of aminoacridines bound to DNA

The binding curves of 1-, 2-, 3-, and 9-aminoacridine and proflavine on native DNA and the circular dichroism (CD) spectra of the bound cations have been determined under the same conditions. The variation of the CD spectra with the amount (r) of aminoacridine bound per DNA phosphorus was of two main kinds: (1) the rotational strength of those aminoacridines which possess a 3-amino group depended markedly on r and decreased to relatively small values (or zero) at zero r; or, (2) the rotational strength changed relatively little with r and tended to a finite value at zero r. The relevance of these observations is discussed with respect to interelation models of the complexes and with respect to possible explanations of the basis of this induction of optical activity.     

Fluorescence decay studies of the DNA-3,6-diaminoacridine complexes

The interaction of several 3,6-diaminoacridines with DNAs of various base composition has been studied by steady-state and transient fluorescence measurements. The acridine dyes employed are of the following two classes: class I - proflavine, acriflavine and 10-benzyl proflavine; class II - acridine yellow, 10-methyl acridine yellow and benzoflavine. It is found that the fluorescence decay kinetics follows a single-exponential decay law for free dye and the poly[d(A-T)]-dye complex, while that of the dye bound to DNA obeys a two-exponential decay law. The long lifetime (tau 1) for each complex is almost the same as the lifetime for the poly[d(A-T)]-dye complex, and the amplitude alpha 1 decreases with increasing GC content of DNA. The fluorescence quantum yields (phi F) of dye upon binding to DNA decrease with increasing GC content; the phi F values for class I are nearly zero when bound to poly(dG) X poly(dC), but those for class II are not zero. This is in harmony with the finding that GMP almost completely quenches the fluorescence for class I, whereas a weak fluorescence arises from the GMP-dye complex for class II. The fluorescence spectra of the DNA-dye complexes gradually shift toward longer wavelengths with increasing GC content. In this connection, the fluorescence decay parameters show a dependence on the emission wavelength; alpha 1 decreases with an increase in the emission wavelength. In view of these results, it is proposed that the decay behavior of the DNA-dye complexes has its origin in the heterogeneity of the emitting sites; the long lifetime tau 1 results from the dye bound to AT-AT sites, while the short lifetime tau 2 is attributable to the dye bound in the vicinity of GC pairs. Since GC pairs almost completely quench the fluorescence for class I, partly intercalated or externally bound dye molecules may play an important role in the component tau 2.     

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.