Michael J. Waring

Complex formation between ethidium bromide and nucleic acids.

The interaction between ethidium bromide and nucleic acids shows a pronounced metachromatic effect which has been used to obtain quantitative data on the process of complex formation. Ethidium binds strongly to both DNA and RNA at sites which appear to be saturated when one drug molecule is bound for every 4 or 5 nucleotides. After the primary sites have been filled, a secondary binding process can occur, leading to the precipitation of a complex containing one drug molecule bound per nucleotide. The strong primary binding to DNA is not influenced by the base-composition or by denaturation of the DNA, but is sensitive to changes in the salt concentration, particularly when magnesium ions are present. Addition of magnesium chloride causes a marked reduction in the strength of the interaction without significantly affecting the number of sites available to bind the drug. The process of complex formation is shown to be reversible in solution by demonstrating an exchange reaction between free and bound ethidium. Complexes between ethidium and DNA can be dissociated by using a cation-exchange resin. The binding of ethidium to DNA follows a similar pattern to that found with proflavine, suggesting that the forces involved in the binding of the two drugs may be similar.

Variation of the supercoils in closed circular DNA by binding of antibiotics and drugs: Evidence for molecular models involving intercalation.

Abstract Intercalation models for the binding of drugs to DNA involve the postulate of drug-induced local uncoiling of the double helix. Such uncoiling results in removal and reversal of the supercoils of closed circular DNA, revealed by changes in the sedimentation coefficient. The effects of 17 substances on the S 20 of φX174 RF † DNA have been tested, with the object of investigating whether changes in the supercoiling of closed circles may be employed to verify intercalative binding. Ethidium bromide alters the supercoiling in the same fashion previously found with other circular DNAs. Equivalence between the supercoils and the accumulated drug-induced uncoiling of the helix occurs at a binding ratio ν c = 0.04 ± 0.008 ethidium molecules bound per nucleotide, from which the number of superhelical turns in φX174 RF is estimated to be −13.3 ± 2.7. Proflavine, hycanthone, daunomycin, nogalamycin, chloroquine and propidium are all believed to intercalate and all affect the supercoils in the same qualitative fashion as ethidium. Non-intercalating substances tested include spermine, streptomycin, methylglyoxal- bis -(guanylhydrazone), berenil, chromomycin and mithramycin; none appear to remove and reverse the supercoiling. LSD and chlorpromazine are similarly without effect. Irehdiamine A is exceptional; it is believed not to intercalate, yet apparent removal and reversal of supercoiling is found. Possible non-intercalative mechanisms to account for uncoiling of the double helix by irehdiamine A are discussed. Actinomycin D affects the supercoils of φX174 RF in a fashion qualitatively and quantitatively comparable with ethidium, providing support for the intercalation model of Muller & Crothers (1968). Different intercalating drugs yield different values of ν c with φX174 RF. These are used to calculate φ, the apparent unwinding angle per bound drug molecule, based upon φ = 12 ° for ethidium (Fuller & Waring, 1964). For four drugs (proflavine, hycanthone, daunomycin and nogalamycin) the calculated values of φ are significantly less than 12 °, yet 12 ° is supposed to be a minimum for an intercalative process. The discrepancy is attributed to the persistence of a certain proportion of the bound drug molecules in a non-intercalated state. Estimates of α, the fraction in the intercalated state, are presented.

Molecular aspects of anticancer drug-DNA interactions

DNA topoisomerases, K. Kohn and R. Ralph the cellular and molecular pharmacology of the anthrapyrazole antitumour agent, D.R. Newell and L.H. Patterson calicheamicin, G.A. Elistad and W.D. Ding molecular pharmacology of intercalator - groove binder hybrid molecules, C. Bailly and J.P. Henichart bleomycins - mechanism of polynucleotide recognition and oxidative degradation, A. Natrajan and S.M. Hecht kinetic analysis of drug-nucleic acid binding modes - absolute rates and effects of salt concentration, W.D. Wilson and F.A. Tanions acridine-based anti-cancer drugs, W.A. Denny and B.C. Baguley the mitomycins - natural cross-linkers of DNA, M. Tomasz.

Supercoiling of polyoma virus DNA measured by its interaction with ethidium bromide

Abstract Exposure of polyoma virus DNA to increasing amounts of the intercalating drug ethidium bromide reduces the sedimentation velocity of the fast component of the DNA (supercoiled molecules) until it is equal to that of the slow component (unsupercoiled molecules). Addition of larger amounts of drug causes the reappearance of a fast component. This is interpreted as being due to the loss and then to the reversal of the supercoiling turns in the DNA molecule. The fact that intercalation of the drug into the molecules causes the loss of supercoiling shows that the supercoiling in the untreated DNA must be due to a deficiency of turns in the Watson—Crick double helix, causing the molecule to take up a conformation with right-handed twists. From the ratio of drug to DNA at the point where supercoiling disappears, it can be calculated that the untreated molecule of polyoma virus DNA has about 12 supercoiling turns. Alternative assumptions concerning the effect of intercalation on the structure of the DNA molecule lead to even higher estimates. Ways in which this amount of supercoiling could be introduced into DNA molecules are discussed.

Structural requirements for the binding of ethidium to nucleic acids

Abstract 1. 1. Ethidium bromide, a phenanthridine drug, forms soluble metachromatic complexes with nucleic acids. The mechanism of the interaction has been investigated by studying the ability of substances related to nucleic acids to shift the visible absorption band of the drug in solution. 2. Purine and pyrimidine nucleotides produce slight shifts to longer wavelengths and the purine compounds are the more effective. However, neither purine nor pyrimidine bases in DNA act as specific binding sites for the drug since complete removal of either type of base from DNA leads to diminished interaction. 3. Although ethidium forms metachromatic complexes with homopolymers, binding curves indicate that the interaction with these materials is quite different from the strong primary binding seen with DNA or RNA. The curves suggest a facilitated binding process which probably represents the weaker secondary binding to nucleic acids. 4. From measurements of the interaction between ethidium and mixtures of homopolymers a correlation is described between the ability of synthetic polynucleotides to bind the drug strongly and the possession of base-paired helical structure. The helical structure need not necessarily be stabilised by purine-pyrimidine contacts. 5. These results provide further evidence that the primary binding of ethidium to nucleic acids occurs by a process of intercalation between adjacent base-pairs while secondary binding occurs by a “stacking” mechanism.

Kinetics of drug-DNA interaction: Dependence of the binding mechanism on structure of the ligand

Abstract Kinetic and equilibrium studies of the binding of several phenanthridines and acridines to DNA have been performed to investigate the physical processes underlying the direct ligand transfer mechanism of drug-DNA interaction· Substitution of the 6-phenyl ring of dimidium with a p -carboxyl residue, or complete removal of either the 6-substituent or the 3-amino group, does not prevent the phenanthridine chromophore from transferring directly between binding sites. Loss of the aromatic ring increases association rate constants three- to ninefold and enhances dissociation rates by factors of up to 12; the rates of direct transfer and dissociation from site 1 are the most perturbed. The presence of a phenyl ring stabilizes the site 1 complex and lowers the binding constant to site 2. Introduction of the p -carboxyl group does not affect the equilibrium distribution of bound forms but produces equivalent increases (2·5-fold) in forward and reverse rate constants for binding to site 1 and for the direct transfer step. The 3-amino group greatly stabilizes the site 1 complex. Its removal accelerates all kinetic processes except for the reverse transfer step; the transfer rate is enhanced 25-fold and binding to site 2 is increased 12-fold. The dissociation rate from site 1 rises by a factor of 45 and that from site 2 by a factor of 5·8. 10-Methyl-9-aminoacridine binds via the direct transfer pathway with rate and equilibrium constants similar to those of the 3-desamino derivative of ethidium. This compound provides the first fully characterized example of an acridine that utilizes bimolecular transfer. By contrast, rivanol (6,9-diamino-2-ethoxyacridine) interacts with DNA via a two-step sequential mechanism analogous to that seen with proflavine, yet its intrinsic association constant is three times higher. This results from tighter ‘external’ attachment to the helix, together with a decrease in equilibrium constant for the insertion step, which is markedly slower than that of proflavine. There appears to be a simple relation between the apparent enthalpy of binding and the number of extracyclic amino substituents on the intercalating chromophore. We propose that the two bound forms that participate in direct ligand transfer represent molecules intercalated via one or other of the grooves of DNA, and that the transfer pathway corresponds to exchange of drug between the wide groove of one helix and the narrow groove of another. The ability to form strongly bound complexes at the surface of the helix appears to play a major role in determining the mechanism of ligand binding.

Kinetics of dissociation of nogalamycin from DNA: comparison with other anthracycline antibiotics

Stopped-flow spectrometry and simple mixing techniques have been employed to investigate the detergent-induced dissociation of anthracycline antibiotics from natural and synthetic DNAs. Both daunomycin and nogalamycin dissociate more slowly from poly(dG-dC) than from poly(dA-dT) but the difference is much more marked for nogalamycin. With an equimolar mixture of poly(dG-dC) and poly(dA-dT), or with poly(dA-dC).poly(dG-dT), dissociation of nogalamycin occurs very slowly. In all cases the release of antibiotic from a synthetic polynucleotide is a one-step process following a single exponential. Dissociation of daunomycin, adriamycin and iremycin from calf thymus DNA is a more complex reaction which requires a two-exponential fit, in contrast to earlier reports, but differences between the behaviour of the three antibiotics are minor. Dissociation of nogalamycin from natural DNA requires a three-exponential fit, is in general far slower, and depends upon the base composition, the level of binding and the time allowed for the complex to equilibrate. It is concluded that sequence selectivity is minimal or lacking for daunomycin, whereas nogalamycin binding is sequence dependent and probably involves migration of the antibiotic between DNA binding sites. There is an inverse correlation between dissociation rate constants and antibacterial potency in simple tests.

Tight binding of the antitumor drug ditercalinium to quadruplex DNA

The structural selectivity of the DNA‐binding antitumor drug ditercalinium was investigated by competition dialysis with a series of nineteen different DNA substrates. The 7H‐pyridocarbazole dimer was found to bind to double‐stranded DNA with a preference for GC‐rich species but can in addition form stable complexes with triplex and quadruplex structures. The preferential interaction of the drug with four‐stranded DNA structures was independently confirmed by electrospray mass spectrometry and a detailed analysis of the binding reaction was performed by surface plasmon resonance (SPR) spectroscopy. The BIAcore SPR study showed that the kinetic parameters for the interaction of ditercalinium with the human telomeric quadruplex sequence are comparable to those measured with a duplex sequence. Slow association and dissociation were observed with both the quadruplex and duplex structures. The newly discovered preferential binding of ditercalinium to the antiparallel quadruplex sequence d(AG3[T2AG3]3) provides new perspectives for the design of drugs that can bind to human telomeres.

Structural perturbations in DNA caused by bis-intercalation of ditercalinium visualised by atomic force microscopy

Atomic force microscopy (AFM) has been used to examine perturbations in the tertiary structure of DNA induced by the binding of ditercalinium, a DNA bis-intercalator with strong anti-tumour properties. We report AFM images of plasmid DNA of both circular and linearised forms showing a difference in the formation of supercoils and plectonemic coils caused at least in part by alterations in the superhelical stress upon bis-intercalation. A further investigation of the effects of drug binding performed with 292 bp mixed-sequence DNA fragments, and using increment in contour length as a reliable measure of intercalation, revealed saturation occurring at a point where sufficient drug was present to interact with every other available binding site. Moment analysis based on the distribution of angles between segments along single DNA molecules showed that at this level of bis-intercalation, the apparent persistence length of the molecules was 91.7 +/- 5.7 nm, approximately twice as long as that of naked DNA. We conclude that images of single molecules generated using AFM provide a valuable supplement to solution-based techniques for evaluation of physical properties of biological macromolecules.

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.