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 micromol. The sharp increase in fluorescence (lambda(em)=530 nm) at 5 micromol of AO was attributed to AO multimer formation. From 0.5 to 5.0 micromol, the AO self-association binding constant (K(assoc)) was determined to be 38620 mol(-1), however, the presence of 150 mmol NaCl increased K(assoc) to 118000 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 micromol DNA ranged from, 32000 mol(-1) at 2 micromol AO, to approximately 3700 mol(-1) at 10 micromol 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 lambda(em) at 530 nm from studies that combine AO, caffeine, and dsDNA.     

De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

Thermal stability of nucleosomes studied in situ by flow cytometry: effect of ionic strength and n-butyrate

Heating of cells permeabilized with ethanol and resuspended in aqueous media increases accessibility of DNA to intercalating dyes such as acridine orange (AO). The curves, representing increase in binding of AO as a function of rise in temperature, indicate that the transitions are cooperative. The transitions are sensitive to ionic strength and occur at lower temperatures when cells are suspended in media of increasing ionic strength. Extraction of histones raises accessibility of DNA to intercalators at room temperature, and heating has little effect on additional binding. The results are interpreted as indicating thermal destruction of nucleosomal structure in nuclear chromatin; dissociation of DNA from core histones results in its increasing ability to intercalate AO, most likely due to increased topological freedom to undergo unwinding and elongation following binding of the intercalator. Preincubation of cells with n-butyrate, known to induce histone hyperacetylation, lowers the heat stability of nucleosomes by about 5 degrees C. On the other hand, no differences are observed between chromatin of mitotic vs interphase cells tested over a wide range of ionic strengths (0.1-0.7 N NaCl). The method appears to be useful as a probe of chromatin structure at the nucleosomal level.     

Comparison of human and mouse sperm chromatin structure by flow cytometry

Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.     

Nuclear chromatin changes during erythroid differentiation of friend virus induced leukemic cells

Friend leukemia (FL) cells grown in the presence of dimethylsulfoxide (DMSO) undergo erythroid differentiation. Acridine orange (AO) binding to DNA and thermal denaturation of DNA in situ were studied in differentiated and non-differentiated FL cells using flow-through cytofluorometry. The differentiated cells bind less AO than do the non-differentiated ones. The difference in AO binding is higher in the spectrum of emission characteristic for AO intercalation (at 530 nm) than for AO stacking (>600 nm) and depends on AO concentration. The difference is abolished after extraction of acid-soluble macromolecules from cells. During stepwise extraction by lowering pH, there is a progressive increase of AO binding to DNA. Most of the increase in AO binding of the non-differentiated cells occurs at pH 2.5-2.0; of the differentiated cells at pH 1.75-1.50. There are differences in sensitivity of DNA in situ to heat-denaturation between differentiated and non-differentiated cells, as evidenced by the variation in height and in position of the melting bands on derivative melting profiles. The changes described suggest that a profound modulation of chromatin structure, perhaps involving altered DNA-histone interactions, occurs during the DMSO-induced erythroid differentiation of FL cells.     

Host-viral effects of chromatin assembly factor 1 interaction with HCMV IE2

Chromatin assembly factor 1 (CAF1) consisting of p150, p60 and p48 is known to assemble histones onto newly synthesized DNA and thus maintain the chromatin structure. Here, we show that CAF1 expression was induced in human cytomegalovirus (HCMV)-infected cells, concomitantly with global chromatin decondensation. This apparent conflict was thought to result, in part, from CAF1 mislocalization to compartments of HCMV DNA synthesis through binding of its largest subunit p150 to viral immediate-early protein 2 (IE2). p150 interaction with p60 and IE2 facilitated HCMV DNA synthesis. The IE2Q548R mutation, previously reported to result in impaired HCMV growth with unknown mechanism, disrupted IE2/p150 and IE2/histones association in our study. Moreover, IE2 interaction with histones partly depends on p150, and the HCMV-induced chromatin decondensation was reduced in cells ectopically expressing the p150 mutant defective in IE2 binding. These results not only indicate that CAF1 was hijacked by IE2 to facilitate the replication of the HCMV genome, suggesting chromatin assembly plays an important role in herpesviral DNA synthesis, but also provide a model of the virus-induced chromatin instability through CAF1.     

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.     

Effect of caffeine and isobutyl-methyl-xanthine on production of binucleate cells and on post-replicative repair.

Caffeine (CAF) but not 3-isobutyl 1-methyl-xanthine (IBMX) displayed a strong DNA anti-repair effect in G2 prophase, as estimated by the frequency of abnormal chromosomes in anaphase-telophase found shortly after treatment. IBMX is more effective than CAF in inhibiting cytokinesis, while the binucleate cells formed by a short treatment with any of these two xanthines have similar cycle kinetics.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytometry (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes fixed by the Boehm-Sprenger procedure optimal for preserving the chromatin pattern. The question was whether fluorochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2 c ratio was still equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the fixation conditions used. The intranuclear and the internuclear SD of the fluorescence intensities describing the fluorescence pattern of the 2c hepatocytes proved to vary according to both the basepair specificity and the binding mode of the fluorochromes. The results reported here argue in favor of an external binding of 7-AMD to DNA and an increased quantum yield of QM when bound to AT-rich DNA. For PI, EB, 7-AMD, and OM, the measured DNA content increased with the fluorescence distribution heterogeneity. This correlation was not observed with other fluorochromes and is suggested to result from decreased fluorochrome accessibility to DNA when the chromatin is condensed. This study demonstrates that under conditions that preserve chromatin organization, only HO for AT-rich DNA and MA for GC-rich DNA can be used, alone or in combination, to measure nuclear DNA content. With other fluorochromes, either the measured DNA content or the chromatin pattern is assessed in suboptimal conditions when fluorescent image cytometry is used.     

The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires the combined activation of histone deposition with the firing of replication origins. We report here the direct interaction of chromatin assembly factor 1 (CAF1), a key factor involved in histone deposition, with the replication kinase Cdc7-Dbf4. We isolated a complex containing both the largest subunit of CAF1 (p150) and the Cdc7-Dbf4 kinase specifically in S phase and thus prove the existence of this interaction in vivo. We then show that the Cdc7-Dbf4 kinase efficiently phosphorylates p150. This event induces a change in p150 oligomerization state, which promotes binding to proliferating cell nuclear antigen (PCNA). Conversely, CAF1 recruitment is reduced in a PCNA/DNA loading assay using Cdc7-depleted extracts. Our data define p150 as a new target for this kinase with implications for the coordination between DNA replication and CAF1 functions.     

From GC-rich DNA binding to the repression of survivin gene for quercetin nickel (II) complex: implications for cancer therapy

The DNA binding and cleavage properties of quercetin nickel (II) complex have been studied, but little attention has been devoted to the relationship between antitumor activity of this complex and DNA-binding properties. In the present study, we report that quercetin nickel (II) complex showed significant cytotoxicity against three tumor cell lines (HepG2, SMMC7721 and A549). Hoechst33258 and AO/EB staining showed HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to quercetin nickel (II) complex. We also demonstrate that the levels of survivin and bcl-2 protein expression in HepG2 cells decreased concurrently, and the levels of p53 protein increased significantly after treatment with quercetin nickel (II) complex by immunocytochemistry analysis. The relative activity of caspase-3 and caspase-9 increased significantly after treatment with the complex. Furthermore, fluorescence measurements and molecular modeling were performed to learn that the complex could be preferentially bound to DNA in GC region. These results imply that quercetin nickel (II) complex may intercalate into the GC-rich core promoter region of survivin, down-regulating survivin gene expression and promoting tumor cells apoptosis. So our results suggest that antitumor activity of quercetin nickel (II) complex might be related to its intercalation into DNA and DNA-binding selectivity, and that the complex may be a promising agent for cancer therapy.     

Chromatin specificity of anti-double-stranded DNA antibodies and a role for Arg residues in the third complementarity-determining region of the heavy chain

A spontaneous, autoreactive autoantibody called SN5-18 (IgG2b, kappa) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vkappa4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vkappa4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vkappa4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of V(H)CDR3 Arg residues in dsDNA binding and the near identity of the SN5-18 sequence to other dsDNA-specific Ab, we tested the contributions of two V(H)CDR3 Arg residues in SN5-18 to chromatin specificity. We found that both these Arg residues at positions 104 and 106 were required for detectable chromatin binding. These results indicate a role for V(H)CDR3 Arg residues in chromatin specificity of lupus-derived autoantibodies. Further, they provide an explanation for a possible discrepancy in the form of tolerance observed in different anti-DNA Ig transgene models.     

Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).     

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).     

Synthesis and structural characterization of piperazino-modified DNA that favours hybridization towards DNA over RNA

We report the synthesis of two C4'-modified DNA analogues and characterize their structural impact on dsDNA duplexes. The 4'-C-piperazinomethyl modification stabilizes dsDNA by up to 5°C per incorporation. Extension of the modification with a butanoyl-linked pyrene increases the dsDNA stabilization to a maximum of 9°C per incorporation. Using fluorescence, ultraviolet and nuclear magnetic resonance (NMR) spectroscopy, we show that the stabilization is achieved by pyrene intercalation in the dsDNA duplex. The pyrene moiety is not restricted to one intercalation site but rather switches between multiple sites in intermediate exchange on the NMR timescale, resulting in broad lines in NMR spectra. We identified two intercalation sites with NOE data showing that the pyrene prefers to intercalate one base pair away from the modified nucleotide with its linker curled up in the minor groove. Both modifications are tolerated in DNA:RNA hybrids but leave their melting temperatures virtually unaffected. Fluorescence data indicate that the pyrene moiety is residing outside the helix. The available data suggest that the DNA discrimination is due to (i) the positive charge of the piperazino ring having a greater impact in the narrow and deep minor groove of a B-type dsDNA duplex than in the wide and shallow minor groove of an A-type DNA:RNA hybrid and (ii) the B-type dsDNA duplex allowing the pyrene to intercalate and bury its apolar surface.     

Quantifying force-dependent and zero-force DNA intercalation by single-molecule stretching

We used single DNA molecule stretching to investigate DNA intercalation by ethidium and three ruthenium complexes. By measuring ligand-induced DNA elongation at different ligand concentrations, we determined the binding constant and site size as a function of force. Both quantities depend strongly on force and, in the limit of zero force, converge to the known bulk solution values, when available. This approach allowed us to distinguish the intercalative mode of ligand binding from other binding modes and allowed characterization of intercalation with binding constants ranging over almost six orders of magnitude, including ligands that do not intercalate under experimentally accessible solution conditions. As ligand concentration increased, the DNA stretching curves saturated at the maximum amount of ligand intercalation. The results showed that the applied force partially relieves normal intercalation constraints. We also characterized the flexibility of intercalator-saturated dsDNA for the first time.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.     

The comparative analysis of interaction between acridine orange and DNA-containing systems]

The interaction between acridine orange (AO) and diluted and concentrated solutions of DNA, DNP systems and chromatin suspension at the physiologic ionic strength was investigated. The effect of AO on DNP systems was also investigated. It was shown that highest possible number of AO molecules bound to DNA made up 70% of the total number of nucleotides. The model of AO binding to DNA is proposed and used for calculation of constants of stronger and weaker AO-binding capacities equal to 6-10(6) M-1 and 1,7-10(5) M-1, respectively. The AO-DNA binding constants in DNP-complex are five as low. The primary number of binding sites in chromatin suspension made up 10% of the corresponding sites in DNA and increased as AO was adsorbed. AO induced the supercontraction of oriented DNP systems at the physiologic ionic strength and the appearance of the low-temperature melting hump.     

Kinetic studies of interaction between acridine orange and DNA

The interaction between acridine orange (AO) and deoxyribonucleic acid (DNA) was studied by the stopped-flow method. The spectral change of AO due to interaction with DNA was followed over the wavelength range 350–600 nm at various concentration ratios of DNA phosphate to dye. The spectral change observed by the stopped-flow method was found distinctly different from that, during the dead-time, leading to a conclusion that the binding of AO to the outside of DNA occurs much faster than the intercalation into base pairs of DNA. The dependence of the rate of reaction on the reactant concentration and on the salt, concentration of the solution was also studied. The results are consistent with the mechanism that the intercalation proceeds via the outside bound state.