Ethidium bromide-dinucleotide complexes. Evidence for intercalation and sequence preferences in binding to double-stranded nucleic acids.

The solution complexes of ethidium bromide with nine different deoxydinucleotides and the four self-complementary ribodinucleoside monophosphates as well as mixtures of complementary and noncomplementary deoxydinucleotides were studied as models for the binding of the drug to DNA and RNA. Ethidium bromide forms the strongest complexes with pdC-dG and CpG and shows a definite preference for interaction with pyrimidine–purine sequence isomers. Cooperativity is observed in the binding curves of the self-complementary deoxydinucleotides pdC-dG and pdG-dC as well as the ribodinucleoside monophosphates CpG and GpC, indicating the formation of a minihelix around ethidium bromide. The role of complementarity of the nucleotide bases was evident in the visible and circular dichroism spectra of mixtures of complementary and noncomplementary dinucleotides. Nuclear magnetic resonance measurements on an ethidium bromide complex with CpG provided evidence for the intercalation model for the binding of ethidium bromide to double-stranded nucleic acids. The results also suggest that ethidium bromide may bind to various sequences on DNA and RNA with significantly different binding constants.     

Ethidium bromide inhibits mitochondrial phosphorylating oxidation.

Ethidium bromide, in addition to combination with mitochondrial nucleic acids, is a phosphorylation inhibitor during glutamate and succinate respiration by mitochondria. Exhaustive washing of ethidium bromide-treated mitochondria did not relieve the inhibition nor significantly decrease the amount of bound dye. Dialysis against a cation exchange resin at 3 degrees for 17 hr removed about 97% of bound dye. This restored phosphorylating capacity to that of untreated mitochondria which had also been dialyzed against the resin. Since state 3 respiration was diminished and state 4 was unaffected by the presence of the acridine dye, and since neither swelling of mitochondria nor release of latent ATPase was observed, then ethidium bromide was not an electron transport inhibitor nor an uncoupler of oxidative phosphorylation. Inhibition of metabolic processes by ethidium bromide may be due in part to depressed generation of mitochondrial ATP.     

Biphasic helix–coil transition of the ethidium bromide·poly(dA-dT) and the propidium diiodide·poly(dA-dT) Complexes. Stabilization of base-pair regions centered about the intercalation site

The nonexchangeable base and sugar proton nmr resonances and the 260 and 278-nm uv-absorbance bands of the nucleic acid were utilized to monitor the temperature-dependent duplex-to-strand transition of the alternating purine–pyrimidine deoxyribopolynucleotide poly(dA-dT) in the absence and presence of ethidium bromide (EB) at phosphate/drug = 50, 28, and 15 and propidium diiodide (PI) at P/D = 50, 25, 15, 10, and 5 in 0.1 M salt between 50° and 100°C. The nmr and optical methods monitor a biphasic duplex-to strand transition for the drug–poly(dA-dT) complexes. We have monitored the dissociation of the drug from the complex at the ethidium bromide phenanthridine ring and side-chain proton nmr resonances and the propidium diiodide 494 and 535-nm uv-absorbance bands and demonstrate that dissociation of the drug corresponds to the higher temperature transition in the biphasic nucleic acid melting curves. The lower temperature cooperative transition is assigned to the opening of drug-free AT base-pair regions in the drug–poly(dA-dT) complex and exhibits an increase in transition midpoint and a decrease in cooperativity with increasing drug concentration. The higher temperature cooperative transition is assigned to the opening of AT base-pair regions centered about the bound drug in the complex and exhibits an increase in the transition midpoint on raising the drug concentration. The large upfield shifts of the phenanthridine ring (but not side chain) protons of ethidium bromide on complex formation demonstrate intercalation of the drug between base pairs of the poly(dA-dT) duplex. The nucleic acid base and sugar resonances of poly(dA-dT) in 0.1 M phosphate undergo chemical shift changes between 0° and 50°C indicative of premelting conformational transition(s).     

A simple method for the fluorescence analysis of nucleic acid-dye complexes in cytological preparations

This communication describes a simple method for recording fluorescence emission spectra of cytological preparations using a conventional fluorescence spectrophotometer. The emission characteristics of "in situ" complexes between some basic fluorochromes (DAPI, 33258 Hoechst, acridine orange, pyronin Y, and ethidium bromide) and nucleic acid containing structures from smears of chicken blood and Ehrlich tumor cells (chromatin, basophilic cytoplasm) are briefly described.     

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.     

The reaction of platinum(II) complexes with DNA. Kinetics of intrastrand crosslink formation in vitro.

The kinetics of the formation of bifunctional DNA platinum(II) adducts (DNA-crosslinks) have been investigated by endonuclease digestion and subsequent HPLC analysis of the soluble nucleotides and nucleotide platinum(II) adducts. The results indicate two waves of crosslinking [rate constants (0.2-0.3) min-1 and (0.015-0.025) min-1] that correlate with changes in ultra violet absorbance and ethidium bromide dependent fluorescence intensity, previously interpreted in terms of two consecutive, local conformational rearrangements of platinum-DNA (Schaller, W., Reisner, H., and Holler, E. (1987) Biochemistry 26, 943-950). The formation of crosslinks at sequences d(GpG) and d(GpNpG) follows identical kinetics. A minimal reaction mechanism is proposed for the binding of cis-diamminedichloroplatinum(II) to DNA under in vitro conditions. The approximately 3-fold higher rate for meso-[1,2-bis(2,6-dichloro-4- hydroxyphenyl)ethylenediamine]diaquaplatinum(II) in comparison to the rate for cis-diamminediaquaplatinum(II) indicates that crosslink formation is affected by the nature of the non-leaving platinum ligand(s).     

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.     

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.     

Characterization of the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II), ethidium bromide and nucleic acids.

The purpose of this study was to characterize the ternary complexes formed in the reaction of cis-diamminedichloroplatinum (II) (cis-DDP) and nucleic acids, in the presence of the intercalating compound ethidium bromide (EtBr). In these ternary complexes, some EtBr is tightly bound to the nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or thin layer chromatography at basic pH. These ternary complexes are formed with double stranded but not with single stranded nucleic acids. They are not formed if cis-DDP is replaced by transdiamminedichloroplatinum(II). The amount of tightly bound EtBr depends upon the sequence of the nucleic acid, being larger with poly (dG-dC).poly(dG-dC) than with poly(dG).poly(dC). Spectroscopic results support the hypothesis that the tight binding of the dye is due to the formation of a bidentate adduct (guanine-EtBr)cis-platin. The visible spectrum of the ternary complexes is blue-shifted as compared to that of EtBr intercalated between the base pairs of unplatinated DNA and it depends upon the conformation of the ternary complex. The fluorescence quantum yield of the ternary complexes is lower than that of free EtBr in water. Tightly bound EtBr stabilizes strongly the B form versus the Z form of the ternary complex poly(dG-dC)-Pt-EtBr and slows down the transition from the B form towards the Z form. The sequence specificity of cis-DDP binding to a DNA restriction fragment in the absence or presence of EtBr is mapped by means of the 3'----5' exonuclease activity of T4 DNA polymerase. In the absence of the dye, all the d(GpG) sites and all the d(ApG) sites but one in the sequence d(TpGpApGpC) are platinated. The d(GpA) sites are not platinated. In the presence of EtBr, some new sites are detected. These results might help to explain the synergism for drugs used in combination with cis-DDP and in the design of new chemotherapeutic agents.     

1H-nuclear magnetic resonance of intercalators and rGCA: a potential mutagenicity probe

Variable temperature 1H-nuclear magnetic resonance (NMR) has been used to study the interaction of the RNA trimer, GpCpA, with the intercalators ethidium bromide and the acridine derivatives; proflavin, 9-amino-acridine, acridine orange, acridine yellow and acriflavin. The complexes formed were studied at nucleic acid to drug ratios of 1:1 and 5:1, the latter being useful in defining the effects of structural variation in the acridine series and in determining the site of intercalation. All the intercalators greatly stabilized the oligonucleotide duplex, the average melting temperature (Tm) increasing by up to 30 degrees C. Significant changes in individual Tms and chemical shifts were observed for all the GpCpA protons. 9-Amino-acridine and acriflavin did not stabilize the GpCpA duplex as substantially as the other acridine derivatives. It is suggested that this intercalator:GpCpA system, and its associated NMR-derived Tm, is a useful physical probe for potential mutagens.     

Rates of platination of -AG- and -GA- containing double-stranded oligonucleotides: effect of chloride concentration

The kinetics of the reactions between 15N-labelled cisplatin and 14-base pair duplex oligonucleotides with either 5'-AG-3' or 5'-GA-3' groupings as the principal platination site are examined in the presence of 60-80 mM chloride by [1H,15N]HSQC 2D NMR spectroscopy. The presence of chloride at these concentrations results in a five-fold decrease in the rate of hydrolysis of cisplatin to cis-[PtCl(NH3)2(OH2)]+ and a two- to twenty-fold decrease in the rate of monofunctional adduct formation. The effects on the rate of closure from monofunctional to bifunctional adducts are less well established but some of these rates appear not to be significantly reduced by the presence of added chloride. The results provide a caution that the use of chloride to quench platination reactions may not be fully effective.     

A helicase assay based on the displacement of fluorescent, nucleic acid-binding ligands.

We have developed a new helicase assay that overcomes many limitations of other assays used to measure this activity. This continuous, kinetic assay is based on the displacement of fluorescent dyes from dsDNA upon DNA unwinding. These ligands exhibit significant fluorescence enhancement when bound to duplex nucleic acids and serve as the reporter molecules of DNA unwinding. We evaluated the potential of several dyes [acridine orange, ethidium bromide, ethidium homodimer, bis-benzimide (DAPI), Hoechst 33258 and thiazole orange] to function as suitable reporter molecules and demonstrate that the latter three dyes can be used to monitor the helicase activity of Escherichia coli RecBCD enzyme. Both the binding stoichiometry of RecBCD enzyme for the ends of duplex DNA and the apparent rate of unwinding are not significantly perturbed by two of these dyes. The effects of temperature and salt concentration on the rate of unwinding were also examined. We propose that this dye displacement assay can be readily adapted for use with other DNA helicases, with RNA helicases, and with other enzymes that act on nucleic acids.     

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.     

Cooperative interactions in the binding of ethidium ion to the self-complementary ribodinucleoside monophosphates CpG and GpC

Complexes exhibiting the characteristics of cooperative interactions are formed by ethidium ion and the self-complementary dinucleoside monophosphates CpG and GpC. Complex formation, observed with an ethidium ion selective electrode, can be described by an equilibrium binding model in which complexes are formed with dinucleoside:ethidium combining ratios of 2:1, 2:2, and 2:3. The total amount of ethidium bound in 2:2 and 2:3 complexes, as calculated from the model, is proportional to a circular dichroism band in CpG-ethidium spectra near 305 nm. Van't Hoff analysis of the model equilibrium constants reveals that the addition of ethidium ion to the 2:1 and 2:2 species is exothermic and that the corresponding entropy changes are large and negative. Cooperative interactions in the binding of ethidium ion and of other ligands to some natural and synthetic polymeric nucleic acids have now been observed in several laboratories, but the present work shows that the effect can arise even with nucleic acid fragments as small as dinucleosides. Apparently, a macromolecular nucleic acid is not essential for cooperative interactions.     

In vitro evolution of cisplatin/DNA monoadducts into diadducts is dependent upon superhelical density.

DNA binding of antitumor platinum(II) compounds accounts for cellular toxicity. Binding of cis-dichlorodiammineplatinum(II) (cis-DDP) to DNA involves the transient presence of monoadducts which evolve in a second phase into difunctional lesions which are far more toxic than the monoadducts. Temporal control of the monoadducts half-live is at least dependent upon the chemical nature of the cis-platinum derivative and the secondary structure of DNA. The effect of the degree of DNA superhelicity on the binding of cis-platinum derivatives as well as on the evolution of monofunctional adducts has been addressed on plasmid DNA. The rate of platination was not affected by the degree of DNA superhelicity. Similarly, when the evolution of the lesions was complete, no variation of toxicity was found with different populations of topoisomers, as determined by bacterial transformation efficiency. In contrast, when the kinetic of difunctional lesions formation was controlled in vitro, we observed a higher rate of formation on a supercoiled plasmid by comparison with a relaxed one. This result suggests that platinum-DNA adduct toxicity could be modulated by the topology of the chromosome.     

Development of a SYBR safe technique for the sensitive detection of DNA in cesium chloride density gradients for stable isotope probing assays

SYBR safe, a fluorescent nucleic acid stain, was evaluated as a replacement for ethidium bromide (EtBr) in cesium chloride (CsCl) density gradients for DNA stable isotope probing (DNA-SIP) assays. The separation of 12C- and 13C-labelled DNA using SYBR safe gave similar results to those obtained using EtBr with pure cultures and environmental samples exposed to a 13C-labelled substrate, while the detection limit of DNA was enhanced by the use of SYBR safe by at least 5 times. The results demonstrated that SYBR safe is a safe, sensitive and effective alternative to the use of ethidium bromide in CsCl density gradients for DNA-SIP assays.     

PMR studies of the self-association of dna intercalating ellipticine derivatives in aqueous solution

In aqueous solution DNA intercalating ellipticine derivatives aggregate in n-mers. The self-association constants K are higher than those of 2-nicthoxy-6-chloro-9-[3-diniethylaminopropyl-amino]-acridine and ethidium bromide. They are of the same order as that of actinomycin D but inferior to that of acridine orange. The increase of the 9-hydroxy-ellipticine constant by addition of sodium chloride shows the importance of anion participation in the mechanism of stacking in accordance with the high energy of self-association. In the stacked n-mets the ellipticine rings aie inverted. The geometry shows the importance of the orientation of the quadiupole axis in the intermotecular association of the intercalating drugs.     

Study of 2.5S RNA of 1,4-alpha-glucan branching enzyme by fluorescent methods using ethidium bromide

The fluorescence yield and lifetime of ethidium bromide complexes with 1,4-alpha-glucan branching enzyme and its free nucleic acid component 2.5S RNA were measured. Both fluorescence parameters showed a 10-fold increase in comparison with those characteristics for the free dye. This increase allows to suggest the existence of double-stranded regions in 2.5S RNA both in the free as well as in the protein bound state. The coefficients of fluorescence polarization were also determined for ethidium bromide complexed with free and protein bound 2.5S RNA. They proved to be 13 and 18% respectively. No concentration depolarization was observed in both types of ethidium bromide and ethidium bromide--enzyme--RNA complexes. This proves that the double-stranded regions are rather short and that two ethidium bromide molecules can't be bound to each of them. The binding isotherms were measured for ethidium bromide absorbed on 2.5S RNA and on the holoenzyme. Their parameters napp and rmax are identical in the cases of free and protein bound 2,5S RNA (rmax = 0.046 +/- 0.001). However the binding constants of ethidium bromide complexes with free and protein bound 2.5S RNA differ significantly (Kapp = 2.2 X 10(6) M-1 for free 2.5S RNA and Kapp = 1.6 X 10(6) M-1 for the holoenzyme). The quantity of nucleotides involved in the two double-stranded regions accessible for ethidium binding is estimated to be about 28%. Increasing of Mg2+ ion concentration up to 10(-3) results in a decrease of ethidium bromide binding with double stranded regions. It may be due to a more compact tertiary structure of 2.5S RNA in the presence of Mg2+ in the free as well as in protein bound state.     

Photolytic binding of the monoazido analog of ethidium to yeast mitochondrial DNA: competition by ethidium.

The [14C]-labeled monoazido analog of ethidium, 3-amino-8-azido-5-ethyl-6-phenylphenanthridinium chloride, when mixed with yeast cells and photolyzed, produced covalent adducts with both nuclear and mitochondrial DNA via the light-generated nitrene. The binding efficiency was about 12 times higher in mitochondrial than nuclear DNA. Moreover, the parent ethidium bromide at a 5-fold excess was an effective competitor for the binding of the monoazide analog with mitochondrial DNA, but not with nuclear DNA.     

Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by intercalating dyes.

The intervening sequence (IVS) excised from the pre-rRNA of Tetrahymena undergoes a self-catalyzed cleavage-ligation reaction to form a covalently closed circular RNA. This cyclization reaction is kinetically inhibited by ethidium bromide (50% inhibition at 22 +/- 14 microM, greater than 99% inhibition at 53 +/- 16 microM for a 20 minute reaction). The dye does not alter the sites of the cyclization reaction, but it does increase the relative amount of reaction at a minor site 19 nucleotides from the 5' end of the IVS. The reversibility of the inhibition and the relative inhibitory strength of acridine orange, ethidium and proflavine suggest that inhibition is due to intercalation of the dye in functionally important secondary or tertiary structures of the IVS. The concentration of dye required to inhibit cyclization is much higher than expected from the known binding constants of such dyes to tRNA. At high Mg2+ to Na+ ratios, conditions which should stabilize RNA structure, a subpopulation of the IVS RNA molecules is resistant to ethidium inhibition, even at 200 microM ethidium. These data are interpreted as reflecting two conformational isomers of the IVS that differ in their reactivity and in their sensitivity to dye binding.