Interaction Of Acridine Orange Research Articles

Spectroscopic, calorimetric and cytotoxicity studies on the combined binding of daunorubicin and acridine orange to a DNA tetrahedron

Chemotherapy–phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.

Multimode binding and stimuli responsive displacement of acridine orange dye complexed with p-sulfonatocalix[4/6]arene macrocycles.

Interaction of acridine orange (AOH+) dye with water soluble anionic p-sulfonatocalix[n]arene (SCXn) hosts, SCX4 and SCX6, having different cavity dimensions, has been investigated using multispectroscopic techniques. Intriguing modulation in the photophysical properties of AOH+ upon interaction with SCXn hosts indicate the formation of different host-guest complexes at different regions of the host concentrations. At lower host concentrations, AOH+ undergoes SCXn assisted aggregation, causing a drastic reduction in fluorescence intensity. At higher host concentrations, aggregated-AOH+-SCXn complexes disintegrate and monomeric-AOH+-SCXn exo and inclusion complexes are eventually formed, leading to a huge fluorescence enhancement finally. Observed effects are more pronounced with SCX6 as compared to SCX4 host. Time-resolved fluorescence studies indicate that at very high host concentrations, there is also a diffusion-controlled dynamic quenching for both monomeric-AOH+-SCXn exo and inclusion complexes, caused by the free SCXn present in the solution, a phenomenon not reported before for such host-guest systems. The aggregated-AOH+-SCXn complexes at lower host concentration were employed to investigate displacement study using an antiviral drug, 1-adamantanamine (AD) and a neurotransmitter, acetylcholine (AcCh), as the competitive binders cum external stimuli, which resulted in a drastic recovery of the fluorescence reduced initially due to aggregation process. Though both the AOH+-SCXn systems act as efficient supramolecular assemblies in sensing AD and AcCh as the analytes through fluorescence "OFF-ON" mechanism, the effect is more pronounced for AOH+-SCX4 system as compared to AOH+-SCX6. SCXn induced interesting modulation in the photophysical properties of AOH+ and the stimulus responsive dye displacement observed for aggregated-AOH+-SCXn systems can expectedly find applications in fluorescence OFF-ON sensing, supramolecular functional materials and similar others.

Acridine orange interaction with DNA: Effect of ionic strength

BackgroundThe study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported. MethodsThe study was performed using steady-state and time resolved optical absorption and florescence, fluorescence correlation spectroscopy and resonant light scattering techniques. ResultsThe presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentration the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggregation. ConclusionsThe interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO efficacy in applications. General significanceThis study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects.

Layer-by-layer films and colloidal dispersions of graphene oxide nanosheets for efficient control of the fluorescence and aggregation properties of the cationic dye acridine orange

Chemically derived graphene oxide (GO) nanosheets have received great deal of interest for technological application such as optoelectronic and biosensors. Aqueous dispersions of GO become an efficient template to induce the association of cationic dye namely Acridine Orange (AO). Interactions of AO with colloidal GO was governed by both electrostatic and π–π stacking cooperative interactions. The type of dye aggregations was found to depend on the concentration of GO in the mixed ensemble. Spectroscopic calculations revealed the formation of both H and J-type dimers, but H-type aggregations were predominant. Preparation of layer-by-layer (LbL) electrostatic self-assembled films of AO and GO onto poly (allylamine hydrochloride) (PAH) coated quartz substrate is also reported in this article. UV–Vis absorption, steady state and time resolve fluorescence and Raman spectroscopic techniques have been employed to explore the detail photophysical properties of pure AO, AO/GO mixed solution and AO/GO LbL films. Scanning electron microscopy was also used for visual evidence of the synthesized nanodimensional GO sheets. The fluorescence quenching of AO in the presence of GO in aqueous solution was due to the interfacial photoinduced electron transfer (PET) from photoexcited AO to GO i.e. GO acts as an efficient quenching agent for the fluorescence emission of AO. The quenching is found to be static in nature. Raman spectroscopic results also confirmed the interaction of AO with GO and the electron transfer. The formation of AO/GO complex via very fast excited state electron transfer mechanism may be proposed as to prepare GO-based fluorescence sensor for biomolecular detection without direct labeling the biomolecules by fluorescent probe.

Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at -0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.

A spectroscopic study of interaction of cationic dyes with heparin

The interaction of two cationic dyes namely, acridine orange and pinacyanol chloride with an anionic polyelectrolyte, heparin, has been investigated by spectrophotometric method.The polymer induced metachromasy in the dyes resulting in the shift of the absorption maxima of the dyes towards shorter wavelengths. The stability of the complexes formed between acridine orange and heparin was found to be lesser than that formed between pinacyanol chloride and heparin. This fact was further confirmed by reversal studies using alcohols, urea and surfactants. The interaction of acridine orange with heparin has also been investigated fluorimetrically.The interaction parameters revealed that binding between acridine orange and heparin arises due to electrostatic interaction while that between pinacyanol chloride and heparin is found to involve both electrostatic and hydrophobic forces. The effect of the structure of the dye in inducing metachromasy has also been discussed.

Fluorescence of acridine orange in the composition of blood and tissues

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or orangelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.

A study of interaction of cationic dyes with anionic polyelectrolytes

The interactions of Acridine Orange with Sodium Alginate and Pinacyanol Chloride with Heparin have been investigated by spectrophotometric method. The polymers induce metachromasy in the dye as evidenced from the considerable blue shift in the absorption maxima of the corresponding dyes. The interaction constant and thermodynamic parameters of polymer-dye interactions have been determined. The effect of additives such as alcohols, and urea on the reversal of metachromasy has been studied. The data has been used to determine the stability of the metachromatic complex and the nature of binding. The thermodynamic parameters of interaction revealed that binding between Acridine Orange and Sodium Alginate involved only electrostatic forces while that between Pinacyanol Chloride involved both electrostatic and hydrophobic forces. The reversal studies using surfactants indicated the involvement of both electrostatic and hydrophobic forces in binding. Based on the results it can be concluded that Pinacyanol Chloride is more effective inducing metachromasy than Acridine Orange.

Interaction of bovine serum albumin with Acridine Orange (C.I. Basic Orange 14) and its sonodynamic damage under ultrasonic irradiation

The interaction of acridine orange (C.I. Basic Orange 14) with bovine serum albumin and the subsequent sonodynamic damage to bovine serum albumin imparted by ultrasonic irradiation were studied. The quenching constant, binding constant and the binding site number were measured using fluorescence quenching. The distance of binding between the dye and the serum albumin was obtained according to Föster's nonradiative energy transfer theory. The effects of ultrasonic irradiation time, dye concentration, pH and ionic strength on bovine serum albumin were determined using UV–vis and fluorescence spectra; the extent of damage was enhanced by a increases in irradiation time, dye concentration and ionic strength, but was reduced by an increase in pH.

Linear sweep polarographic determination of nucleic acids using acridine orange as a bioprobe

The interaction of acridine orange (AO) with double-stranded (ds) DNA in aqueous solution was investigated by linear sweep polarography (LSP) on a dropping mercury working electrode (DME). In pH 2.5 Britton-Robinson (B-R) buffer solution, AO had a sensitive linear sweep polarographic reductive peak at -0.89 V (vs. SCE), which could be greatly inhibited by the addition of dsDNA, with a positive shift of the peak potential. Based on the decrease of the reductive peak current, a new quantitative electrochemical determination method for dsDNA was developed with a linear range of 2.0?20.0 mg l-1 and the linear regression equation: ?Ip" (nA) = 111.90 C (mg l-1)+125.32 (n = 9, ? = 0.997). The influences of commonly co-existing substances, such as metal ions, amino acid, etc., on the determination were also investigated. The method is sensitive, rapid and simple with good selectivity. The new proposed method was further applied to the detection of RNA and three synthetic samples containing dsDNA with satisfactory results. The binding number and the equilibrium constant between dsDNA and AO were calculated by an electrochemical method.

Fluorometric determination of DNA using nano-SiO2 particles as an effective dispersant and stabilizer for acridine orange

A sensitive fluorometric method for the determination of ctDNA (calf thymus DNA) is presented. It has good selectivity and sensitivity and uses nano-SiO2 particles as an effective dispersant and stabilizer for acridine orange (AO). Compared to resonance light scattering (RLS) and the conventional method that uses organic dyes as fluorescence probe, the new method is more tolerant towards coexisting foreign substances and also more stable. With 20 mg nano-SiO2 particles, 10 µmol L−1 AO, at pH 8.01 and an ionic strength of 0.02 mol L−1, the interaction of AO with nano-SiO2 and ctDNA results in fluorescent signal enhancement. The extent of enhancement was in good proportion to the concentration of ctDNA at excitation/emission wavelengths of 490/523 nm, respectively. The calibration curve was linear over 0.66–55.60 µg mL−1. The determination limit (3σ) was 15 ng mL−1. The method was applied to the determination of ctDNA in synthetic samples with satisfactory results.

Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels

Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 μM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 °C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0±4.29, 15.6±1.48 and 1.06±0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.

Study of the interaction of nucleic acids with acridine orange-CTMAB and determination of nucleic acids at nanogram levels based on the enhancement of resonance light scattering

The resonance light scattering (RLS) of acridine orange (AO) are greatly enhanced by both nucleic acid and cetyltrimethyl ammonium bromide (CTMAB). Based on this, nucleic acids can be sensitively determined. The interaction of AO with nucleic acid in the presence of CTMAB is studied by using RLS, absorption spectroscopy, zeta potential assay, transmission electron microscope (TEM) image and molecular molding. On its surface, nucleic acid acts as the template to promote the assembly of both AO and CTMAB in close proximity, which formatted large AO-nucleic acid-CTMAB association. Therefore, RLS intensity of cationic dye is greatly enhanced.

Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations >2 μmol. The sharp increase in fluorescence (λ em=530 nm) at 5 μmol of AO was attributed to AO multimer formation. From 0.5 to 5.0 μmol, the AO self-association binding constant ( K assoc) was determined to be 38 620 mol −1, however, the presence of 150 mmol NaCl increased K assoc to 118 000 mol −1 attributed to the charge neutralization. The K assoc for AO with CAF was confirmed to be 256 mol −1. K assoc for the binding of AO with 20 μmol DNA ranged from, 32 000 mol −1 at 2 μmol AO, to approximately 3700 mol −1 at 10 μmol AO, in the absence of NaCl. This AO concentration dependency of K assoc value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at λ em at 530 nm from studies that combine AO, caffeine, and dsDNA.

CD and UV absorption bandshapes of DNA—acridine orange complexes: Influence of the interactions of purine—pyrimidine base pairs with dyes in intercalated model complexes

Induced CD and UV absorption spectral bandshapes of DNA—acridine orange (AO) complexes have been calculated for a model in which DNA intercalates one AO dye between every other base pair ( P/D=4), using the linear response polarizability theory of copolymer systems. The spectra show a strong dependence on the intercalated positions and the orientational angles of the AO dyes in the DNA pocket. The base pair sequence effect in the bandshapes which reflects the interaction of AOs with the DNA bases can clearly be observed, particularly in the ultraviolet region. It is necessary to take into account the interactions of DNA bases with AOs in electronic excited states in order to calculate reliable induced CD and UV absorption spectral bandshapes.

Mechanism of acridine orange interaction with phospholipids and proteins in renal microvillus vesicles.

The mechanism of interaction of acridine orange (AO), a fluorescent, weak base, with rabbit kidney brush border membrane vesicles (BBMV) has been studied by absorption, and steady-state and time-resolved fluorescence spectroscopy. Equilibrium binding experiments indicate that AO binds to an apparent single class of sites on BBMV with a dissociation constant of 90 microM and site stoichiometry of 810 nmol/mg protein. The absorption spectra AO indicate that BBMV induces aggregation of AO; experiments with lipid vesicles show that the aggregation requires BBMV membrane proteins. Fluorescence stopped-flow experiments in which 0.15 mg/ml BBMV is mixed with increasing concentrations of AO result in a time course of fluorescence enhancement for [AO] less than 1.5 microM, and of fluorescence quenching for [AO] greater than 1.5 microM. Similar stopped-flow experiments with phosphatidylcholine lipid vesicles result only in a fluorescence enhancement time course. These results indicate the presence of two parallel pathways for AO binding to BBMV: one for AO binding to BBMV lipid, the other for AO binding to BBMV protein. Nanosecond lifetime measurements and fluorescence titration experiments confirm the presence of two environments for AO in BBMV. Fluorescence stopped-flow experiments indicate that AO responds to the imposition of an outwardly directed proton gradient by a rapid (less than 0.5 s) decrease in fluorescence, corresponding to re-equilibration of AO into the acidic intravesicular compartment, followed by an increase in fluorescence, corresponding to proton flux across the membrane. These findings have been incorporated into a stepwise mechanism for AO interaction with BBMV which have direct implications for the use of AO as a pH indicator in biological systems.

Interaction of acridine orange with various ligands

The behaviour of acridine orange in the presence of various ligands, in aqueous and non-aqueous solution, is illustrated on the basis of some recent experimental results. The interaction of acridine orange with cyclodextrins is discussed in some more details.

A comparison by ultracentrifugation of the effects on DNA of ethidium bromide and of acridine orange at low ionic strength

The application of scaled particle theory to the gels formed by DNA in the ultracentrifuge has provided values for the effective length and the effective radius of the DNA particle. Ethidium bromide has been shown to cause extensive lengthening of the DNA in dilute salt. Acridine orange interaction with DNA resulted in modest changes in DNA dimensions. These results are explained in terms of binding for acridine orange and of denaturation of DNA by ethidium bromide.

Interactions of acridine orange with double stranded nucleic acids. Spectral and affinity studies.

Spectral properties of acridine orange (AO) alone or in complexes with natural and synthetic nucleic acids of various base composition have been studied in aqueous solutions by absorption and fluorescence spectroscopy. The dimerization constant and absorption spectra of the dye in monomeric and dimeric form were established; dimerization of AO resulted in quenching of its fluorescence. Complexes of the dye with synthetic nucleic acids differed in the degree of enhancement of fluorescence quantum yield, varying between 1.42 to 2.38 fold as compared to AO monomer; these differences, however, were not base-dependent. Affinity of the dye to natural and synthetic polymers was studied and analyzed using McGhee-von Hippel model of polymer-ligand interactions. Because the sterical requirement for intercalative binding assumes interaction of dye monomer, the correction for AO dimerization was made in all calculations. All studied DNAs (natural and synthetic ones, the latter being homopolymer pairs or alternating copolymers of A,T or G,C or I,C base composition) had similar intrinsic association constants (KI = 5 X 10(4) - 1 X 10(5), M-1) and binding site size (n = 2.0-2.4 b.p.). The exception was poly(dA).poly(dT), having KI = 1.2 X 10(4) and n = 19.3 b.p. The results of KI measurement for calf thymus DNA and AO in different sodium ion concentration were in good agreement with predictions of the counterion condensation theory. The intercalation of AO into DNA is discussed in view of recent theoretical models of DNA-ligand interactions.

Interactions of acridine orange with nucleic acids: Properties of complexes of acridine orange with single stranded ribonucleic acid

Interactions between acridine orange (AO) and nucleic acids (calf thymus DNA, and homoribo- and homodeoxyribo-polynucleotides) were studied in solutions containing ethanol as a cosolvent. Light absorption, scattering and luminescence were measured as a function of AO concentration at different dye/phosphate (D/P) ratios, and the data were analyzed using the McGhee-von Hippel probabilistic model of the polymer-ligand interactions. The absorption spectra of AO complexes with four homoribopolymers are presented. The intrinsic association constants and cooperativity coefficients of the formation of the complexes were calculated. The effects of ethanol (up to 35%, v/v) on these interactions were concentration dependent and may be extrapolated to zero concentration of this cosolvent. The possibility of destabilization of the double helix of nucleic acids by AO at high D/P ratios is discussed in light of the available thermodynamic data.