Measurement of the expected DNA lengthening caused by mono-and bisintercalating drugs using electron microscopy

PM₂ DNA molecules were treated with intercalating reagents (ethidium bromide, ethidium dimer, acridine dimer) and observed by electron microscopy. The adaptation of different electron microscopy techniques has enabled the determination of DNA lengthening upon drug intercalation. A 50% length increase was generally obtained for DNA saturated with the drugs. This result is in agreement with the intercalation model proposed by Lerman. In some cases (ethidium dimer), an increase of length larger than 50% can be obtained. Experimental conditions of DNA spreading strongly interfere with the DNA–drug interaction. In some cases it was possible to estimate the apparent binding constants and also to distinguish the mono- from the bisintercalating derivatives in their reaction with DNA.     

Linear dichroism and polarized fluorescence of dye-complexed DNA fibers

Summary A simple method to obtain well orientated DNA fibers for studying the ordered binding of dyes and fluorochromes by linear dichroism and polarized fluorescence is described. The metachromatic dye toluidine blue and the intercalating fluorochromes ethidium bromide and acridine orange showed a perpendicular alignement to DNA; the minor groove binding fluorochromes 33258 Hoechst and DAPI appeared parallel. Thus, DNA fibers represent a suitable cytochemical test substrate for studying the orientation of bound dyes by polarization methods.     

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.     

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.     

Spectroscopic and molecular modeling studies of caffeine complexes with DNA intercalators.

Recent studies have demonstrated that caffeine can act as an antimutagen and inhibit the cytoxic and/or cytostatic effects of some DNA intercalating agents. It has been suggested that this inhibitory effect may be due to complexation of the DNA intercalator with caffeine. In this study we employ optical absorption, fluorescence, and molecular modeling techniques to probe specific interactions between caffeine and various DNA intercalators. Optical absorption and steady-state fluorescence data demonstrate complexation between caffeine and the planar DNA intercalator acridine orange. The association constant of this complex is determined to be 258.4 +/- 5.1 M-1. In contrast, solutions containing caffeine and the nonplanar DNA intercalator ethidium bromide show optical shifts and steady-state fluorescence spectra indicative of a weaker complex with an association constant of 84.5 +/- 3.5 M-1. Time-resolved fluorescence data indicate that complex formation between caffeine and acridine orange or ethidium bromide results in singlet-state lifetime increases consistent with the observed increase in the steady-state fluorescence yield. In addition, dynamic polarization data indicate that these complexes form with a 1:1 stoichiometry. Molecular modeling studies are also included to examine structural factors that may influence complexation.     

Interaction of novel tris-intercalators with DNA. Spectrofluorometric studies.

Novel DNA binding ligands (1 and its stereoisomer 2) which contain three potentially intercalating units in a linear molecular skeleton were prepared. From a study of the displacement of ethidium bromide from several natural and synthetic polynucleotides, both compounds 1 and 2 were found to show an AT base-pair preference in the interaction with DNAs with 2 a slightly higher affinity for DNA. This result is in sharp contrast to that for acridine and anthraquinone, because these two compounds exhibit a GC-preference.     

Ethidium bromide (EB) binding to nucleosomal DNA : Effects on DNA cleavage patterns

Binding of ethidium bromide (EB) to chromatin DNA induces structural changes in nucleosomes. The characteristic cleavage patterns of nucleosomal DNA after digestion with either micrococcal nuclease or pancreatic deoxyribonuclease are altered in the presence of the intercalating dye. Instead, apparently random digestion occurs. Polylysine reduces the amount of EB-binding sites in nucleosomal DNA. Since the intercalation of EB is known to proceed from the minor groove of DNA, polylysine supposedly occupies the same site of the nucleosomal DNA moiety.     

DNA intercalators induce specific release of HMG 14, HMG 17 and other DNA-binding proteins from chicken erythrocyte chromatin.

Chicken erythrocyte nuclei were incubated with DNA intercalating agents in order to isolate from chromatin specific DNA-binding proteins whose binding specificity may be determined by DNA secondary and/or tertiary structure. The intercalating agents ethidium bromide (EtBr) and propidium iodide induce the specific release of high mobility group proteins HMG 14 and HMG 17 under low ionic strength conditions. Chloroquine (CQ) intercalation also results in the selective liberation of HMG 14 and HMG 17, but, in addition, selectively releases other nuclear proteins (including histone H1A) in a pH- and ionic strength-dependent fashion. The use of this new 'elutive intercalation' technique for the isolation and purification of 'sequence-specific' and 'helix-specific' DNA-binding proteins is suggested.     

Initial studies of a phytoestrogen-deoxyribonucleic acid interaction

Molecular modeling studies show that estrogens such as estradiol complement the topography of spaces between base pairs in unwound DNA and simultaneously hydrogen bond phosphate moieties on opposite strands. We demonstrate here that the phytoestrogen coumestrol has this capability, in addition to its documented properties of UV absorbance at lambda greater than 300 nm and fluorescence. The latter properties enable spectroscopic examination of interactions with DNA by methods not possible with estrogenic steroids. On exposure to calf thymus DNA, the UV spectrum of coumestrol displays a bathochromic shift and simultaneous hypochromic effect with an isosbestic point at 370 nm, suggesting a shift between coexisting free and bound states. Similar results are observed with the intercalating agents adriamycin, ethidium bromide, and acridine. The fluorescence spectrum of coumestrol is quenched on exposure to DNA as are those of adriamycin and acridine. Coumestrol differs from the intercalators in that denatured DNA does not affect its UV spectrum or alter its relative fluorescence yield. Unlike classical intercalators, coumestrol has no influence on the thermal stability of calf thymus DNA. Preliminary electrophoretic analysis of DNA plasmid conformers indicates that coumestrol is incapable of significantly altering DNA superhelical density, in contrast to ethidium bromide. These initial physicochemical data provide evidence for the DNA base-estrogen electronic and/or hydrophobic interactions suggested by modeling studies, yet tend to rule out classical intercalation as an explanation for these phenomena.     

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.     

Effect of ligand binding on the buoyant density of DNA in Nycodenz gradients.

The effect of ligand binding upon the buoyant density of DNA in Nycodenz gradients has been studied using DNAs of differing base compositions. The effect of both intercalating ligands (ethidium bromide and proflavin) and non-intercalating ligands (distamycin A, DAPI and netropsin) has been studied. The binding of intercalating ligands to DNA has essentially no effect on the buoyant density of DNA in Nycodenz gradients. The non-intercalating ligands were found to increase the buoyant density of DNA in a base specific manner. The increase in buoyant density can be interpreted in terms of disruption of the hydration shell of the DNA molecule caused by the binding of the ligand along the minor groove of the DNA helix.     

Modulation of platinum antitumor drug binding to DNA by linked and free intercalators

We report the DNA binding site preferences of the novel molecule AO-Pt, in which the anticancer drug dichloro(ethylenediamine)platinum(II) is linked by a hexamethylene chain to acridine orange. The sequence specificity of platinum binding was mapped by exonuclease III digestion of 165 and 335 base pair restriction fragments from pBR322 DNA. Parallel studies were carried out with the unmodified anticancer drugs cis-diamminedichloroplatinum(II) (cis-DDP) and dichloro(ethylenediamine)platinum(II), [Pt(en)Cl2]. Oligo(dG) sequences are the most prevalent binding sites for AO-Pt, with secondary binding occurring mainly at d(AG) sites. cis-DDP and [Pt(en)Cl2] bind less readily to the secondary sequences, with cis-DDP showing greater binding site selectivity than [Pt(en)Cl2]. The DNA intercalator ethidium bromide promotes binding of [Pt(en)Cl2] and cis-DDP to many sites containing d(CGG) and, to a lesser extent, d(AG) sequences. AO-Pt exhibits enhanced binding to these sequences without the need for an external intercalator. Unlinked acridine orange, however, does not promote binding of [Pt(en)Cl2] and cis-DDP to d(CGG) and d(AG) sequences. These results are discussed in terms of the sequence preferences, stereochemistry, and relative residence times of the intercalators at their DNA binding sites. By modulating local structure in a sequence-dependent manner, both linked and, in the case of ethidium, free intercalators can influence the regioselectivity of covalent modification of DNA by platinum antitumor drugs.     

The effects of ethidium bromide and Mg++ ion on strand exchange in the Hin-mediated inversion reaction

The biochemical reaction of a site-specific recombinase such as Hin invertase or gammadelta resolvase starts with binding of the recombinase to its recombination site and cleavage of the DNA in the center of the site. This is followed by strand exchange and finally ligation of the ends of the recombined strands. Previous biochemical studies have shown that Hin invertase and gammadelta resolvase cannot proceed beyond DNA cleavage in the absence of Mg++ ion, indicating that these recombinases require Mg++ ion in the strand exchange process. We have observed that the intercalating agent, ethidium bromide (2 microM), does not interfere with DNA cleavage, but slows strand exchange in a concentration-dependent manner. Levels of Mg++ ion below 5 mM also slow strand exchange substantially. We infer that random intercalation of ethidium bromide inhibits unwinding of the double helix at the recombination site in the negatively supercoiled DNA and propose that Mg+ may be required for Hin to deform the secondary structure of B-DNA prior to strand exchange.     

The DNA and RNA specificity of eilatin Ru(II) complexes as compared to eilatin and ethidium bromide

Eilatin-containing ruthenium complexes bind to a broad range of different nucleic acids including: calf thymus (CT) DNA, tRNA(Phe), polymeric RNAs and DNAs, and viral RNAs including the HIV-1 RRE and TAR. The nucleic acid specificity of Lambda- and Delta-[Ru(bpy)2eilatin]2+ have been compared to that of the 'free' eilatin ligand, and to the classic intercalating agent ethidium bromide. Interestingly, all four compounds appear to bind to nucleic acids by intercalation, but the trends in nucleic acid binding specificity are highly diverse. Unlike ethidium bromide, both eilatin and the eilatin-containing coordination complexes bind to certain single-stranded RNAs with high affinity (K(d) < or = 1 microM). Eilatin itself is selective for electron-poor polymeric purines, while the eilatin-coordination complexes exhibit preference for the polypyrimidine r(U). These results show how the binding specificity of an intercalating ligand can change upon its incorporation into an octahedral metal complex.     

Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells

Friend leukemia cells from exponentially growing or differentiated (DMSO-induced) cultures were permeabilized and their DNA was stained with 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33342, acridine orange, ethidium bromide, propidium iodide, quinacrine, 7-amino-actinomycin D, mithramycin, or chromomycin A3. Accessibility of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with 0.1N HCl. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HCl there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7-amino-actinomycin D (13-fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.     

An evaluation of DNA fluorochromes, staining techniques, and analysis for flow cytometry. I. Unperturbed cell populations

Several preparative techniques (detergent treatment, ethanol fixation, and hypotonic cell lysis), DNA fluorochromes, and methods of numerical analysis (planimetric or curve-fitting) were compared for the estimation of cell-cycle kinetic parameters (G1, S, G2 + M) by flow cytometry. In addition, coefficients of variation (CV), relative fluorescence, and G1/chicken erythrocyte (CRBC) ratios were measured and the effects of the proportion of cycling cells and cellular RNA content were examined. DNA fluorochromes were ranked by relative fluorescence: 4,6-diamidino-2-phenylindole > ethidium bromide/mithramycin > Hoechst 33342 > mithramycin > ethidium bromide > acridine orange approximately equal to propidium iodide. The first four (DNA-specific stains) gave lower CVs than the remainder (DNA intercalators). Detergent treatment also increased relative fluorescence and slightly lowered CVs. Comparable results were obtained for the kinetic parameters independently of stain or staining procedure; intercalating dyes with cells of a high RNA content not treated with RNAse and acridine orange being the exceptions. Of the two methods of numerical analysis, the planimetric technique was more consistant. Although highly consistant G1/CRBC ratios were obtained for any one stain, independently of staining procedures, variations between stains were noted. It is suggested that the detergent treatment in combination with DNA-specific stains provide optimal results.     

Binding of ethidium and bis(methidium)spermine to Z DNA by intercalation

The interaction of ethidium bromide, a DNA intercalating drug, and bis( methidium )spermine, a DNA bis-intercalating compound, with the left-handed Z form of poly(dG-dC) has been studied in 4.4 M NaCl. Spectrophotometric analysis using absorption, fluorescence and circular dichroism indicates that the complex formed between ethidium and Z DNA resembles very closely that formed with B DNA. This suggests that ethidium binds to Z DNA by intercalation. 31P NMR spectra are presented showing both the conversion of the Z form to the B form with increasing amounts of drug and the typical Z form spectrum at low binding densities. Data are also presented which show that the bifunctional intercalator bis( methidium )spermine binds to Z DNA in a manner similar to its binding to B DNA, i.e., by bis-intercalation. These results are important for our understanding the behavior of Z DNA and its biological significance.     

Characterization of the DNA-binding properties of the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin

The DNA-binding properties of the receptor for 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) were investigated using chromatography on DNA-cellulose columns. A maximal binding of about 40% of the total receptor complex to DNA-cellulose was observed. In order to interact with DNA, the receptor must first bind TCDD. A heat-activation step followed by gel permeation chromatography using Sephadex G-25 increased the binding of the cytosolic receptor to DNA. The DNA-binding ability of the receptor was almost lost following mild proteolysis using trypsin or alpha-chymotrypsin, although these treatments did not reduce its ligand binding capacity and had no apparent effect on its size. Furthermore, pre-treatment of the DNA-cellulose column with an intercalating drug, ethidium bromide, resulted in inhibition of the binding of the TCDD-receptor complex to DNA, indicating that not only electrostatic interactions but also the configuration of DNA are of importance in receptor-DNA interactions.     

An evaluation of the conformational changes in the superhelical DNA of eukaryotic cells by direct nucleoid fluorometry. II. The characteristics of the change in acridine orange fluorescence in studying the superhelical DNA of rat thymocytes

Comparative studies of acridine orange (AO) and ethidium bromide interactions with supercoiled DNA (scDNA) of thymocytes were performed in which various conformational changes were induced. AO may be efficiently used for evaluation of conformational alterations of scDNA. Moreover, employing the maximum values of AO fluorescence allows to determine the maximum levels of scDNA relaxation.     

A comparison of fluorochromes for direct viable counts by image analysis

Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4',6-diamidino-2-phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.