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.     

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 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.     

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.     

Interaction of phenosafranine with nucleic acids and model polyphosphates: II. Characterisation of phenosafranine binding to DNA

The binding of phenosafranine (PS) to DNA was studied by a combination of spectroscopic methods (absorption and fluorescence) together with hydrodynamic measurements (sedimentation and viscosity), Analysis of spectroscopic binding curves revealed that the strength of binding of PS to DNA is generally lower than that of proflavine. These measurements enabled recognition of several modes of interaction between PS and native DNA: strong monomer binding prevailing at high DNA phosphate/dye ratios (p) comprising binding outside the DNA helix as well as intercalation; two modes of dimer binding at lower values of p; and probably also weak surface-binding of monomers as p approaches unity. Longer surface-bound aggregates of PS were not detected because of the low tendency of the dye to form aggregates, though the presence of dimeric species distinct frorn pure surface-stacked PS dimer was indicated by various observations. It occurs over a broad range of p values Starting at p ≈110 for ionic strengths 10^?3–10^?1. Thermal denaturation data indicate that this species is bound more strongly than pure surface-bound stacked dimer. Its dimeric character may be explained in terms of interaction of an intercalated dye molecule with an adjacent outside-bound one as suggested for acridines by Armstrong et al. Various properties of this species are discussed. Both strong and weak modes or binding of PS to DNA are sensitive to the presence of organic solvents. The effectiveness of solvents to destabilise the complexes substantially coincides with their capacity to alter the water activity. Viscometric investigations reveal that in the region of strongest bindins (p ? 15) the elongation of the DNA helix by approximately 0.18 nm per bound PS molecule is accompanied by a strong negative change in persistence length, i.e. bending. Similar bending is also found at higher levels of binding (p ? 15) induced by less lightly bound PS molecules, in which region, however, the unusually high elongation of approximately 0.34 nm per bound PS molecule is observed.     

Anthracycline-binding induced DNA stiffening, bending and elongation; stereochemical implications from viscometric investigations

Upon interaction of the three anthracycline antibiotics daunomycin, adriamycin, and aclacinomycin A with calf thymus DNA the relative changes of both DNA contour length, delta L/Lo, and persistence length, delta a/ao, have been determined as a function of r, the ratio of bound ligand molecules per DNA mononucleotide. From the r dependence of delta a/ao a measure for the stiffening effect and also the angle gamma of ligand-induced DNA bending could be derived. Experimental basis are titration viscometric measurements upon both low and high molecular weight DNA. It was found that the DNA contour length increases linearly with r by approximately 0.34 nm per bound drug molecule. The comparatively very high DNA stiffening effect measured in solution is understandable as a result of helix clamping by at least two anthracycline groups of sufficient long distance. The variation of gamma on DNA interaction with different anthracycline derivatives find their explanation in terms of different values of the mismatch to in-register binding prior to complex formation. From an analogous interpretation of viscosity measurements by Arcamone and coworkers upon high molecular weight DNA with many anthracycline derivatives it can be concluded that DNA interaction by both amino sugar and 9-acetyl group are responsible for the generation of strong anthracycline binding mediated DNA stiffening effects in solution. (A combined analysis of the viscosity measurements by Cohen & Eisenberg and Armstrong et al. upon DNA interaction with proflavine indicates a very small DNA stiffening effect, gamma = 6.7 sigma and a helix elongation by 0.35 nm per bound ligand molecule.)     

Sedimentation studies on the interaction of proflavine with deoxyribonucleic acid

A method is described of using photography to measure the concentrations of a small ligand (proflavine) above and below the boundary of a macromolecule (DNA, both native and denatured) sedimenting in the ultracentrifuge. The measurements are used to determine the extent of the binding of proflavine to DNA, and the results compared with those obtained by a spectrophotometric method. The results obtained by the two methods agree within 10%, thus validating the spectrophotometric technique under these conditions. The variation of the sedimentation coefficient with the extent of binding of proflavine was also studied. The results are discussed in relation to previously observed changes in the viscosity of the solutions.     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Spectroscopic studies on the interaction mechanisms of safranin T with herring sperm DNA using acridine orange as a fluorescence probe

Under the condition of physiological pH environment (pH = 7.40), the interactions of safranin T (ST) with herring sperm DNA were studied by means of spectral methods using acridine orange (AO) as a fluorescence probe. The spectroscopic characteristics of DNA–AO in the case of ST (along with the increase of concentration) were observed in an aqueous medium. The binding constants for ST stranded DNA and competitive bindings of ST interacting with DNA–AO systems were examined by fluorescence spectra, and the binding mechanism of ST with DNA was researched via viscosity measurements. All the testimony manifested that bonding modes between ST and DNA were evidenced to be intercalative binding and electrostatic binding, and the combining constant of ST with DNA was obtained. The binding of ST to DNA was driven by entropy and enthalpy through the calculated thermodynamic parameters (Δ_rH_m^?, Δ_rS_m and Δ_rG_m^?). Copyright © 2014 John Wiley & Sons, Ltd.     

Altered physiochemical properties of the deoxyribonucleic acid-mitomycin C complex. Evidence for the conformational change in deoxyribonucleic acid

Binding of the antibiotic mitomycin C to sonicated calf thymus DNA results in increased viscosity and an unaltered sedimentation constant of DNA. Flow dichroism measurements of the mitomycin C-DNA complex indicate that the 310-nm absorbance vector of the chromophore of the bound drug is oriented at approximately 57.2 degrees relative to the helix axis. A conclusion drawn from these results is that mitomycin C does not intercalate between base pairs, but rather, it is bound in one of the grooves. Binding of mitomycin C causes a number of changes which are DNA size dependent: (1) increased viscosity of sonicated, decreased viscosity of nonsonicate DNA; (2) unaltered sedimentation rate of sonicated, increased rate of nonsonicated DNA; (3) reduced electrophoretic mobility of nonsonicated DNA; (4) electron microscopic appearance of sonicated DNA-mitomycin complexes which is similar to that of control, while nonsonicated DNA complexes which display highly coiled, looped structures not seen in control nonsonicated DNA. These size-dependent effects are interpreted as indicative of conformational distortion of DNA at rare intervals, caused by a minor fraction of total bound mitomycin. The parallel used of sonicated and nonsonicated DNA as probes for certain effects of drug binding may be useful for detecting this type of phenomenon in general.     

Kinetic and hydrodynamic studies of the complex of proflavine with poly A·poly U

Relaxation kinetic experiments reveal general similarity between the mechanism of binding of proflavine to poly A·poly U and DNA. There are differences in detail, however. For example, the rate constants are roughly an order of magnitude smaller for the former, and the thermodynamic parameters of the individual steps are also different. The total heat and free energy for intercalation of free dye are quite similar in the two cases. As was the case with DNA, considerable dye (up to 25% of the bound form) is attached externally to the double helix, even in the strong binding region of the isotherm. Sedimentation measurements on small, rodlike fragments of poly A·poly U reveal a length increase on binding proflavine of a magnitude similar to that found with DNA. This length increase seems to become smaller under conditions (high temperature) where the relaxation measurements indicate a higher fraction of externally bound dye.     

Interaction of proflavine and 9-aminoacridine with DNA at temperatures below and above the melting temperature

The influence of temperature on the binding of 9-aminoacridine and of proflavine to E. coli DNA in 10^?3M NaCl solution has been determined by a spectrophotometric technique. The inadequacy of the expression normally used for the determination of the extent of binding is discussed with reference to measurements at temperatures above which dissociation of the double helix occurs. A method of determining the relative extents of binding to native and denatured DNA at elevated temperatures is described.     

Interaction of metal ions with nucleic acids and related compounds. II. Studies on Au(III)-nucleic acid system

The nature of interaction of Au(III) with nucleic acids was studied by using methods such as uv and ir spectrophotometry, viscometry, pH titrations, and melting-temperature measurements. Au(III) is found to interact slowly with nucleic acids over a period of several hours. The uv spectra of native calf-thymus DNA 9pH 5.6 acetate buffer containing (0.01M NaCIO₄) showed a shift in λ max to high wavelengths and an increase in optical density at 260 nm. There was a fourfold decrease in viscosity (expressed as η_sp/c). The reaction was faster at pH 4.0 and also with denatured DNA (pH 5.6) and whole yeast RNA (pH 5.6). The order of preference of Au(III) (as deduced from the time of completion of reaction) for the nucleic acids in RNA > denatured DNA > DNA. The reaction was found to be completely reversible with respect KCN. Infrared spectra of DNA-Au(III) complexes showed binding to both the phosphate and bases of DNA. The same conclusions were also arrived at by melting-temperature studies of Au(III)-DNA system. pH titrations showed liberation of two hydroxylions at r = 0.12 [r = moles of HAuCl₄ added per mole of DNA-(P)] and one hydrogen ion at r = 0.5. The probable binding sites could be N(1)/N(7) of adenine, N(7) and/or C(6)O of guanine, N(3) of cytosine and N(3) of thymine. DNAs differing in their (G = C)-contents [Clostridium perfingens DNA(G = C, 29%), salmon sperm DNA (G + C, 42%) and Micrococcus lysodeikticus DNA(G + C, 29%), salmon sperm DNA (G = C, 72%)] behaved differently toward Au(III). The hyperchromicity observed for DNAs differing in (G + C)-content and cyanide reversal titrations indicate selectivity toward ( A + T)-rich DNA at lw values of r. Chemical analysis and job's continuous variation studies indicated the existence of possible complexes above and below r = 1. The results indicate that Au(III) ions probably bind to hte phosphate group in the initial stages of the reaction, particularly at low values of r, and participation of the base interaction also increases. Cross-linking of the two strands by Au(III) may take place, but a complete collapse of the doulbe helix is not envisaged. It is probable that tilting of the bases or rotaiton of the bases around the glucosidic bond, resulting in a significant distrotion of the double helix, might take place due to binding of Au(III) to DNA.     

Effect of Mg++ and polyamines on proflavine binding to T2 DNA

Proflavine binding may be used as a probe of the environment and interactions of DNA. In this paper we report the effects of the divalent cations Mg⁺⁺ and putrescine and the trivalent cation spermidine on the proflavine–Na DNA binding equilibrium. Difference spectroscopy at 430 nm was used to determine apparent proflavine–DNA binding constants K at several concentrations of each cation for temperatures between 15 and 43°C, and at a constant total ionic strength of 0.1M. Mg⁺⁺, putrescine, and spermidine all have greater effects on K than expected on the basis of ionic strength alone in the order spermidine > Mg⁺⁺ ? putrescine. van't Hoff analysis of K(T) enabled calculation of ΔH° and ΔS°, which are affected differently by each cation. These differences are discussed qualitatively in terms of such concepts as release of condensed counterions, localized or unlocalized condensation, hydration, and restriction of molecular and internal rotation.     

Kinetic and equilibrium studies of the interactions of silver ions with poly(A)

The interaction of silver ions with poly(A) was studied by potentiometric titration, uv spectrophotometry, and stopped-flow spectroscopy. For 0 < r_b < 0.5, where r_b is moles of silver ion bound per mole of nucleotide base, there exists only one type of binding for poly(A). Using McGhee's theory, the binding parameters, such as intrinsic binding constant, number of sites per nucleotide, and cooperativity, were determined from the potentiometric titration data. Using the stopped-flow method, one relaxation time was observed in 0 < r₀ < 0.5, where r₀ is the moles of silver ions added per mole of nucleotide base. The concentration dependences of the relaxation time suggest that the binding of silver ions to poly(A) proceeds through the following mechanism: where M is free silver ions, P the free binding sites on poly(A), and C and C′ are two forms of the complex. The nature of the binding of silver ions to poly(A) is also discussed.     

Semiflexibility of collagens in solution

The persistence length of lugworm cuticle collagen in 0.1M acetic acid was evaluated as 1600 ～ 1800 Å by Yamakawa-Fujii's model for a wormlike chain from the sedimentation constant and the intrinsic viscosity. The persistence length was further examined for a series of sample “collagen sonicates” produced by varying the duration of sonic irradiation. To estimate the salt effect on the persistence length, measurements were made over a range of NaCl concentrations from 0 to 0.1M. The results showed that the cuticle collagen and collagen sonicates had identical values of persistence length and that the neutral salt effect for the cuticle collagen was far smaller than that for DNA.     

93 Spectroscopic investigation of methylene blue binding to DNA

The interaction of methylene blue (MB) with DNA has been investigated by UV absorption spectra, Fluorescence spectra and UV-melting method. Analysis of the results of the melting experiments shows that melting temperature (T _m) of the complexes increases with the [total ligand]: DNA ratio (r) at two concentrations of Na⁺ (2?mM Na⁺ and 20?mM Na⁺) providing support for conclusion that MB is a stabilizer of DNA helix structure. By contrast, the shapes of dependences of width of transition (ΔT) on r at low and high [Na⁺] are different which points to the existence of different types of binding modes of MB with DNA. UV-spectroscopy experiments and fluorescence spectra indicated that the binding modes of MB with DNA depended on r. At high r (r?>?0.25), remarkable hypochromic effect with no shift of λ _max in the absorption spectra of MB was observed. The fluorescence of MB was quenched which indicated that MB was bound to phosphate groups of DNA by electrostatic interaction. At low r ratios (r?<?0.2), the absorption spectra of MB upon increasing the concentration of DNA showed gradually decrease in the peak intensities with a red shift. This phenomenon is usually associated with molecular intercalation into the base stack of the ds-DNA. Using the Scatchard’s model, the complex formation constants for MB with DNA were determined: the binding constant K?≈?6.5?×?10⁵ and binding site size n?≈?4. Obtained data are not typical for intercalation model of ligands to DNA. Moreover, comparison between these data and our early experimental results of interaction of ethidium bromide with DNA made it possible to suggest that this binding type of MB is, more probably, semi-intercalation mode (Vardevanyan et al., 2003). This conclusion is in accordance with the analysis of the model structures of MB–DNA complexes which clearly shows the importance of solvent contributions in suggested structural form (Tong et al., 2010).     

Dielectric behavior of DNA-proflavine complex

The dielectric relaxation of namtive DNA and DNA–proflavine complexes at different DNA phosphate (P) to dye (D) ratios, were investigated in the frequency range 100 c/sec to 100 Kc/sec. The proflavine molecules were found to have a profound effect on the static dielectric constant and the relaxation time of the polymers. The static dielectric constant was oberserved to decrese with increasing level of added proflavine. At P/D = 1, the variation of dielectric constant with frequency was small. Relaxation time (τ) was greater for the DNA–proflavine complexes compared to that for free DNA, Maximum value of the relaxation time was obtained at P/D = 10. The increase in the relaxation time and decrease in the static dielectric constant were attributed to the increase in length and meutralization of surface charges of the DNA molecules, respectively, as aresult of proflavine binding.     

Kinetics of drug-DNA interaction. Dependence of the binding mechanism on structure of the ligand

Kinetic and equilibrium studies of the binding of several phenanthridines and acridines to DNA have been performed to investigate the physical processes underlying the direct ligand transfer mechanism of drug-DNA interaction· Substitution of the 6-phenyl ring of dimidium with a p-carboxyl residue, or complete removal of either the 6-substituent or the 3-amino group, does not prevent the phenanthridine chromophore from transferring directly between binding sites. Loss of the aromatic ring increases association rate constants three- to ninefold and enhances dissociation rates by factors of up to 12; the rates of direct transfer and dissociation from site 1 are the most perturbed. The presence of a phenyl ring stabilizes the site 1 complex and lowers the binding constant to site 2. Introduction of the p-carboxyl group does not affect the equilibrium distribution of bound forms but produces equivalent increases (2·5-fold) in forward and reverse rate constants for binding to site 1 and for the direct transfer step. The 3-amino group greatly stabilizes the site 1 complex. Its removal accelerates all kinetic processes except for the reverse transfer step; the transfer rate is enhanced 25-fold and binding to site 2 is increased 12-fold. The dissociation rate from site 1 rises by a factor of 45 and that from site 2 by a factor of 5·8.10-Methyl-9-aminoacridine binds via the direct transfer pathway with rate and equilibrium constants similar to those of the 3-desamino derivative of ethidium. This compound provides the first fully characterized example of an acridine that utilizes bimolecular transfer. By contrast, rivanol (6,9-diamino-2-ethoxyacridine) interacts with DNA via a two-step sequential mechanism analogous to that seen with proflavine, yet its intrinsic association constant is three times higher. This results from tighter ‘external’ attachment to the helix, together with a decrease in equilibrium constant for the insertion step, which is markedly slower than that of proflavine. There appears to be a simple relation between the apparent enthalpy of binding and the number of extracyclic amino substituents on the intercalating chromophore.We propose that the two bound forms that participate in direct ligand transfer represent molecules intercalated via one or other of the grooves of DNA, and that the transfer pathway corresponds to exchange of drug between the wide groove of one helix and the narrow groove of another. The ability to form strongly bound complexes at the surface of the helix appears to play a major role in determining the mechanism of ligand binding.     

Characterization of the binding of neomycin/paromomycin sulfate with DNA using acridine orange as fluorescence probe and molecular docking technique

The binding of neomycin sulfate (NS)/paromomycin sulfate (PS) with DNA was investigated by fluorescence quenching using acridine orange (AO) as a fluorescence probe. Fluorescence lifetime, FT-IR, circular dichroism (CD), relative viscosity, ionic strength, DNA melting temperature, and molecular docking were performed to explore the binding mechanism. The binding constant of NS/PS and DNA was 6.70 × 10³/1.44 × 10³ L mol^?1 at 291 K. The values of ΔH^θ, ΔS^θ, and ΔG^θ suggested that van der Waals force or hydrogen bond might be the main binding force between NS/PS and DNA. The results of Stern–Volmer plots and fluorescence lifetime measurements all revealed that NS/PS quenching the fluorescence of DNA–AO was static in nature. FT-IR indicated that the interaction between DNA and NS/PS did occur. The relative viscosity and melting temperature of DNA were almost unchanged when NS/PS was introduced to the solution. The fluorescence intensity of NS/PS–DNA–AO was decreased with the increase in the ionic strength. For CD spectra of DNA, the intensity of positive band at nearly 275 nm was decreased and that of negative band at nearly 245 nm was increased with the increase in the concentration of NS/PS. The binding constant of NS/PS with double-stranded DNA (dsDNA) was larger than that of NS/PS with single-stranded DNA (ssDNA). From these studies, the binding mode of NS/PS with DNA was evaluated to be groove binding. The results of molecular docking further indicated that NS/PS could enter into the minor groove in the A–T rich region of DNA.