Interaction Of Acridine Orange Research Articles

Denaturation of RNA and DNA in situ induced by acridine orange

The products of interaction of acridine orange (AO) with single-stranded (ss) nucleic acids are precipitates which exhibit red luminescence. Titration of rRNA or thymus DNA with AO results in formation of such products suggesting that the dye, per se, denatures double-stranded (ds) sections of these biopolymers. This transition, measured as the increase of red luminescence, a concomitant decrease of green fluorescence, and followed by an increase of light scatter of the AO-nucleic acid complexes, is cooperative and at 0.15 N NaCl occurs at 4–20 and 10–50 μM range of AO concentration for rRNA and DNA, respectively. The changes in stainability of nucleic acids in situ, in permealized cells, occur at higher AO concentration. Thus, the transition of RNA in situ is biphasic and seen at 20–120 μM AO. In the presence of EDTA, however, the change is monophasic and shifted to the 10–30 μM range of AO concentration. The change in stainability of DNA also shows two phases: one at 30–60 μM and another at 70–120 μM of AO. Extraction of basic proteins with 0.08 N HCl shifts the transition of DNA to the 30–60 μM AO concentration and makes it monophasic. The observed differences in denaturability of RNA vs DNA explain the specificity of AO in differential staining of these bipolymers in histochemical reactions. In living cells the products of interaction of AO with nucleic acids are detected by electron microscopy. In the cytoplasm of interphase cells the formation of dense precipitates within ribosomes and polysomes, simultaneous with a specific retraction of ribosome-polysome complexes from the periphery of the cell to the nucleus is evident. The latter suggests higher order organization of these particles involving their association with each other or with the nucleus via polyanionic macromolecules which collapse upon binding with AO. The DNA in heterochromatin is more sensitive to AO-induced denaturation, as evidenced by the fact that the dense complexes are formed preferentially in the regions of condensed chromatin of the interphase nucleus, or in metaphase chromosomes.

Spectrophotometric investigation on the interaction of acridine orange with methylene blue, phenosafranine, and disulphine blue in aqueous medium

The results of dye–dye interactions for the pairs, acridine orange – methylene blue, acridine orange – phenosafranine, and acridine orange – disulphine blue have been presented. It has been found that at comparable concentrations the interaction is rather weak, and the formation of mixed entities is only possible at relatively high proportions of one of the components. However, the evidence of mixed dimers in the form of a new absorption band is lacking. Phenosafranine remarkably promotes dimerization of acridine orange. An experimental demonstration of the dimer spectra of acridine orange has thus been possible. The interaction of the anionic dye disulphine blue with cationic acridine orange has been observed to be the weakest of all the pairs, indicating the vital role of the structural parity for interaction. At comparable low concentrations, none of the dye-pairs studied have revealed compound metachromasy in the presence of a polyelectrolyte, suggesting unfavorable mixed dye aggregation under ordinary conditions.

Thermodynamics of mucopolysaccharide–dye binding. III. Thermodynamic and cooperativity parameters of acridine orange–heparin system

AbstractBinding isotherms for acridine orange (AO)–heparin systems can be evaluated solely on the basis of quantitative fluorescence spectroscopic measurements. The evaluation of thermodynamic parameters indicates that the interactions of AO with heparins from several animal sources are similar to each other in magnitude. Binding is highly exothermic (ΔH = −6 kcal mol−1) and is stabilized by dye–polymer and dye–dye (coopertive) interactions, as well as by entropic factors (ΔS = +7 e.u.). The predominant stabilizing factor appears to be the electrostatic attraction between the AO cation and the heparin polyanion, although the other factors are important as well. At 24°C the value of the cooperative binding constants for the various heparins range from 8.8 to 11.3 × 105M−1, corresponding to a free energy of −8 kcal mol−1. The degree of cooperativity, which is a direct measure of dye–dye interaction, varies with polymer:dye ratio; the theoretical basis for this variation remains to be elucidated. Electrophoretic data indicate that each heparin sample consists of a mixture of species, each with its own charge density. This precludes definitive interpretation of observed small differences in the values of the thermodynamic parameters among the various samples until each sample can be resolved into its components.

Interaction of acridine orange and polyanions: fluorimetric determination of binding strengths and the influence of simple electrolytes

Binding affinities of Acridine Orange and six polyanions have been determined by measuring association constants in essentially salt-free solution, and also by observing competition between the dye and a simple salt for the polyanion site. Differences were found in the dye-binding order for the six polyanions using the two techniques. These differences were related to the breakdown in the uniform binding behaviour of carboxy and sulphate groups bound to the same polymer backbone as the salt concentration is increased. A mechanism is proposed which fits the thermodynamic parameters calculated by the application of Schwarz treatment and the Langmuir isotherm. The difference in the free-energy of binding between the strongest binding polymer, polystyrene sulphonate (ΔG°=–41 kJ mol–1) and the weakest binding, hyaluronic acid (ΔG°=–29 kJ mol–1) could be accounted for by considering coulombic site-dye interactions which destabilise the dye aggregates. The main influence promoting dye binding appears to be the free-energy of dye aggregation.The degree of co-operativity of dye binding, q, has also been determined. An attempt has been made to relate this parameter to the physical properties of the polyanion chain. It appears unlikely that q has the significance initially attached to it.

Interaction of acridine orange and sodium poly-α, L-glutamate

The interaction of acridine orange and sodium poly-α, L-glutamate in the pH range of 3.8–6.8 was studied by a spectral method and viscosity measurement. From the binding data, the molar change in entropy, enthalpy and the number of binding sites on polymer were calculated. The values of ΔS° were positive. ΔS° for binding decreasing extensively in pH range of 5.0. These results are explained by the transition of helix ⇋ coil of the polymer. The intrinsic viscosity reduction in the presence of the dye, in pH 3.8 is due to the formation of dye bridges between the polymer helix.

Spectroscopic properties of complexes of acridine orange with glycosaminoglycans. I. Soluble complexes.

AbstractThe interaction of acridine orange with dermatan and chondrotin sulfates results in the formation of complexes containing bound dye molecules ordered into dissymmetric arrays. Complexes containing an excess of available disaccharide residues compared to dye are completely soluble, and exhibit biphasic circular dichroism bands. Analysis of the dependence of the extrinsic circular dichrosim on dye aggregation indicates the presence of extended dye stacks bound to the glycosaminoglycan. Complexes formed in solutions containing an excess of dye are only partially soluble, and exhibit circular dichroism spectra having band shifts and intensity changes relative to the soluble complexes. The latter complexes show a sharp drop in induced circular dichroism with temperature, due to a cooperative change in the structure of the complex. The structural order of the dye–glycosaminoglycan complex may differ from the intrinsic structure of the glycosaminoglycan itself in dilute solution.

Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry

Secondary structure of RNA was studied in human leukemic SK-L7 cells by their staining with acridine orange (AO) and fluorescence measurements in a flow-through cytofluorimeter. Parallel measurements of RNAse-treated cells made it possible to distinguish and to measure separately those components of total cell fluorescence that are due to AO interaction with RNA. Like DNA, double helical and single-stranded RNA in situ stain differentially with AO. Hence, the extent of RNA in double helical conformation, interacting with AO by intercalation, can be measured. In 10 −3M phosphate buffer, RNA in situ denatures at 40–60 °C. Mg ions stabilize RNA against thermal denaturation; EDTA markedly decreases stability of RNA in double helical conformation. A slow, progressive denaturation of RNA in situ is seen as a result of cell exposure to AO at low ionic strength at room temperature. It is presumed that the changes described reflect mostly alterations of secondary structure of rRNA. Results of our in situ studies conform with data of others on rRNA denaturation in intact isolated ribosomes. This suggests that the ribosome anchoring in cytoplasm is not associated with double helical rRNA regions. Cell staining with AO under conditions of full RNA denaturation and in the absence of DNA denaturation offers differential, simultaneous staining of RNA and DNA. This approach in cell staining provides a new parameter for automated cell classification based on both quantity and conformation of RNA in situ.

Interaction of acridine orange and amylose in a binary liquid system dimethyl sulphoxide-water

Interaction of acridine orange and amylose in a binary liquid system dimethyl sulphoxide-water

Some differences between 470 nm and 510 nm in the acridine-orange-sensitized photodynamic actions on yeast cells

The two wavelengths that correspond to the absorption of two complexes formed in the interactions of acridine orange (AO) with nucleic acids were different in a few aspects of photodynamic action on yeast, Saccharomyces cerevisiae. ( 1) The dose-survival curve at 470 nm is not the same in shape as that at 510 nm. ( 2) The efficiency for the induction of gene conversion at 510 nm is higher than that obtained at 470 nm on the basis of both dose and survival.

Binding and interaction of acridine orange with intraphage DNA

The interaction of acridine orange with intraphage DNA is weak, also perhaps the binding is little, although sufficient time is allowed in order that binding may reach equilibrium. The equilibrium dye-binding increases with the rise of temperature and becomes optimum at a temperature of 50–55° which is about 25–30° lower than the temperature of total DNA release and melting. Studies of interaction of acridine orange and ethidium bromide to intact phage indicate that, although the pahge DNA may be accessible for binding to both acridine orange and ethidium bromide, the binding is temperature specific for acridine molecules and perhaps involves at least two kinds of interaction. By contrast the interaction of ethidium bromide is by only one mode ( i.e. intercalation) and increases continuously with the rise of temperature. The phage DNA in situ has been found to be hyperchromic relative to native DNA in vitro to an extent (hyperchromicity 6 %) similar to that reported for T 2 phage and gives rise to considerable polarization of fluorescence when oriented. The temperature specific binding of acridine orange to phage DNA seems to arise as a result of some competition between bindings of weaker interaction and stronger interaction until an equilibrium between the two bindings is established. The latter may be controlled by the tight configuration and non-native conformation of the intraphage DNA. The interaction or binding is not likely to be determined by any selective permeability of the phage envelope.

Kerr constant dispersion. 3. The interaction of acridine orange with DNA.

Kerr constant dispersion. 3. The interaction of acridine orange with DNA.

The Interaction of Acridine Orange and Proflavine with Polyadenylic Acid1

The Interaction of Acridine Orange and Proflavine with Polyadenylic Acid¹

INTRAVITAL FLUORESCENCE MICROSCOPY IN THE QUALITATIVE EVALUATION OF THE INTERACTION OF ACRIDINE ORANGE WITH NUCLEIC ACID IN INTACT LIVING HUMAN ORGANS.

OBSERVATIONS on living organs excited by ultra-violet irradiations have been almost entirely confined to work on experimental animals. The specific fluorescence method was largely developed through the work of Ellinger and Hirt1,2, who termed it intravital fluorescence microscopy. The method found use in the investigation of resorptive and excretory functions of different organs, and offered valuable information about the physico-chemical conditions and processes in living cells, in particular the electrostatic phenomena. It also helped in the elucidation of various problems concerning the function of kidneys3,4, liver5,6, blood vessels7, and nerves8, and other organs of experimental animals.

The interaction of deoxyribonucleic acid and acridine orange

The interaction of acridine orange (AO) and deoxyribonucleic acid (DNA) in acetate, citrate, and phosphate buffers between pH 3.15 and 7.0 was studied spectrophotometrically and fluorometrically. The absorbancy of the dye was decreased and the fluorescence quenched in the presence of DNA, and further addition of DNA caused a shift of the absorption maximum to longer wavelengths and an enhancement of fluorescence. The point of wavelength shift in absorbancy and maximum quenching of fluorescence occurred at the same weight concentration of DNA, namely 10 −2 mg. DNA/ml. of reaction mixture. These results are interpreted as indicating the formation of two distinct complexes between DNA and acridine orange.