Binding of quinoline analogues of echinomycin to deoxyribonucleic acid. Role of the chromophores.

Two novel antibiotics were isolated, designated compounds 1QN and 2QN respectively, having quinoline rings in place of one or both of the quinoxaline chromophores of echinomycin. Each removes and reverses the supercoiling of closed circular duplex DNA from bacteriophage PM2 in the fashion characteristic of intercalating drugs. For compound 1QN, the unwinding angle at I0.01 is almost twice that of ethidium, whereas for compound 2QN the value is indistinguishable from that of ethidium. Binding of both analogues produced changes in the viscosity of sonicated rod-like DNA fragments corresponding to double the helix extension found with ethidium, a feature characteristic of bifunctional intercalation by quinoxaline antibiotics. These results suggest that both compounds 1QN and 2QN behave as bifunctional intercalators but that compound 2QN produces only half the helix unwinding seen with compound 1QN and the natural quinoxalines. Binding curves for the interaction of both analogues with a variety of synthetic and naturally occurring nucleic acids were determined by solvent-partition analysis. Values for compound 2QN were also obtained by a fluorimetric method and found to agree well with the solvent-partition measurements. Compound 1QN bound most tightly to Micrococcus lysodeikticus DNA and, like echinomycin, exhibited a broad preference for (G + C)-rich DNA species. For compound 2QN no marked (G + C) preference was indicated, and the tightest binding among the natural DNA species studied was found with DNA from Escherichia coli. The two analogues also displayed different patterns of specificity in their interaction with synthetic nucleic acids. Compound 2QN bound to poly(dA-dT) slightly more tightly than to poly-(dG-dC), whereas compound 1QN displayed a large (approx. 11-fold) preference in the opposite sense. There was evidence of co-operativity in the binding to poly(dA-dT). It may be concluded that the chromophore moieties play an active role in determining the capacity of quinomycin antibiotics to recognize and bind selectively to specific sequences in DNA.     

Interaction between synthetic analogues of quinoxaline antibiotics and nucleic acids. Changes in mechanism and specificity related to structural alterations.

The interaction with DNA of six chemically synthesized derivatives of the quinoxaline antibiotics was investigated. Five of the compounds bound only weakly to DNA or not at all; for these substances spectrophotometric measurements, sedimentation studies with closed circular duplex bacteriophage-PM2 DNA and thermal-denaturation profiles were used to determine limits fot the binding constants. No interaction could be detected with two products of degradation of echinomycin (quinomycin A), one of which, echinomycinic acid dimethyl ester, had the lactone linkages opened, whereas the other retained an intact octapeptide ring but had a broken cross-bridge. The other compounds studied were des-N-tetramethyl-triostin A ('TANDEM') and its derivatives. A derivative of 'TANDEM' IN WHICH benzyloxycarbonyl moieties replace both quinoxaline chromophores had binding constants to nucelic acids in the range 10(2)--10(3)-1, whereas no interaction could be detected for a benzyloxycarbonyl derivative that, in addition, had the cross-bridge broken. The derivative of 'TANDEM' with L-serine in place of D-serine in both positions showed no detectable interaction with Clostridium perfringens DNA, whereas the binding constant to poly(dA-dT) was approx 2 X 10(3)M-1. 'TANDEM' itself bound strongly to DNA, and the bathochromic and hypochromic shifts in its u.v.-absorption spectrum in the presence of DNA were similar to those seen with echinomycin. From the effect on the sedimentation coefficient of closed circular duplex bacteriophage-PM2 DNA the mechanism of binding was shown to involve bifunctional intercalation, typical of the naturally occurring quinoxaline antibiotics. Solvent-partition analysis was used to determine binding constants for the interaction between 'TANDEM' and a variety of natural and synthetic DNA species. The pattern of specificity thus revealed differed markedly from that previously found with the naturally occurring quinoxaline antibiotics. Most striking was the evident large preference for (A + T)-rich DNA species, in complete contrast with echinomycin and triostin A. The highest binding constant was found for poly(dA-dT), the interaction with which appeared highly co-operative in character. The conformations adopted by those quinoxaline compounds that bind strongly to DNA were examined withe aid of molecular models on the basis of results derived from n.m.r. and computer studies. It appears that the observed patterns of base-sequence specificity are determined, at least in part, by the structure and conformation of the sulphur-containing cross-bridge.     

Functional cross-talk between fatty acid synthesis and nonribosomal peptide synthesis in quinoxaline antibiotic-producing streptomycetes

Quinoxaline antibiotics are chromopeptide lactones embracing the two families of triostins and quinomycins, each having characteristic sulfur-containing cross-bridges. Interest in these compounds stems from their antineoplastic activities and their specific binding to DNA via bifunctional intercalation of the twin chromophores represented by quinoxaline-2-carboxylic acid (QA). Enzymatic analysis of triostin A-producing Streptomyces triostinicus and quinomycin A-producing Streptomyces echinatus revealed four nonribosomal peptide synthetase modules for the assembly of the quinoxalinoyl tetrapeptide backbone of the quinoxaline antibiotics. The modules were contained in three protein fractions, referred to as triostin synthetases (TrsII, III, and IV). TrsII is a 245-kDa bimodular nonribosomal peptide synthetase activating as thioesters for both serine and alanine, the first two amino acids of the quinoxalinoyl tetrapeptide chain. TrsIII, represented by a protein of 250 kDa, activates cysteine as a thioester. TrsIV, an unstable protein of apparent Mr about 280,000, was identified by its ability to activate and N-methylate valine, the last amino acid. QA, the chromophore, was shown to be recruited by a free-standing adenylation domain, TrsI, in conjunction with a QA-binding protein, AcpPSE. Cloning of the gene for the QA-binding protein revealed that it is the fatty acyl carrier protein, AcpPSE, of the fatty acid synthase of S. echinatus and S. triostinicus. Analysis of the acylation reaction of AcpPSE by TrsI along with other A-domains and the aroyl carrier protein AcmACP from actinomycin biosynthesis revealed a specific requirement for AcpPSE in the activation and also in the condensation of QA with serine in the initiation step of QA tetrapeptide assembly on TrsII. These data show for the first time a functional interaction between nonribosomal peptide synthesis and fatty acid synthesis.     

The use of radiolabelled triostin antibiotics to measure low levels of binding to deoxyribonucleic acid.

Triostin antibiotics, which contain a cyclic peptide with a disulphide bridge, have been prepared by growing Streptomyces triostinicus in the presence of inorganic [35S]-sulphate. The labelled triostin A has been shown to behave in all respects similarly to the authentic natural product and to enable a much more sensitive radiochemical adaptation of the solvent-partition method for determining antibiotic binding to DNA. By this means, binding isotherms at low, biologically relevant levels (down to one antibiotic molecule per gene) have been measured. The results indicate the existence of some tight binding sites in natural DNA species that are preferentially occupied at low concentrations. No evidence has been found for any allosteric transitions provoked by interaction between these antibiotics and natural DNA species, though there is evidence for co-operativity in the binding of triostin A to poly(dA-dT). For the first time accurate isotherms have been determined for the binding of triostin C to DNA; its binding constants for a variety of polydeoxynucleotides are uniformly tighter than those of triostin A but fall into the same ranking order when different species of natural DNA are compared.     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

A solvent-partition method for measuring the binding of drugs to DNA. Application to the quinoxaline antibiotics echinomycin and triostin A.

The development of a novel solvent-partition method for measuring the interaction between nucleic acids and drugs of limited water solubility is described. Factors relevant to the choice of a suitable water-immiscible solvent are summarised. i-Amyl acetate was selected for studying the binding of echinomycin and triostin A to DNA. Details of the experimental determination of extinction and partition coefficients are given; in the i-amyl acetate/buffer system employed for most experiments, the partition coefficients for echinomycin and triostin A were 111 +/- 4 and 943 +/- 23, respectively. Equilibration of echinomycin between the organic and aqueous phases was 90% complete within a few minutes, and a period of 2 h shaking was found satisfactory to ensure full attainment of equilibrium. Representative results are presented showing specific binding of the quinoxaline antibiotics to DNA, strong preference for double-helical as opposed to heat-denatured or single-stranded DNA, and restricted uptake by closed circular duplex PM2 DNA. The method is potentially applicable, with appropriate modifications, to the study of interactions between other ligands and DNA.     

DNA-binding affinities and sequence selectivity of quaternary benzophenanthridine alkaloids sanguinarine, chelerythrine, and nitidine

A comparative study on the intercalating binding of sanguinarine, chelerythrine, and nitidine with CT DNA, poly(dG-dC).poly(dG-dC), poly(dA-dT).poly(dA-dT), and seven sequence-designed double-stranded oligodeoxynucleotides has been performed using fluorometric and spectrophotometric techniques, aiming at providing insights into their sequence selectivity for DNA-binding. The results show that both sanguinarine and nitidine bind preferentially to DNA containing alternating GC base pairs [d(TGCGCA)(2)], while chelerythrine exhibits quite distinct sequence selectivity from sanguinarine, which shows a high specificity for DNA containing contiguous GC base pairs [5'-TGGGGA-3'/3'-ACCCCT-5'].     

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 Å for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentration. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6·10⁶ M^?1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behaviour cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala³, Ala⁷]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.     

The different binding modes of Hoechst 33258 to DNA studied by electric linear dichroism.

The binding mode of the bisbenzimidazole derivative Hoechst 33258 to a series of DNAs and polynucleotides has been investigated by electric linear dichroism. Positive reduced dichroisms were measured for the poly(dA-dT).poly(dA-dT)- and poly(dA).poly(dT)-Hoechst complexes in agreement with a deep penetration of the drug into the minor groove. Similarly, the drug displays positive reduced dichroism in the presence of the DNAs from calf thymus, Clostridium perfringens and Coliphage T4. Conversely, negative reduced dichroisms were obtained when Hoechst 33258 was bound to poly(dG-dC).poly(dG-dC), poly(dA-dC).poly(dG-dT) and poly(dG).poly(dC) as well as with the GC-rich DNA from Micrococcus lysodeikticus indicating that in this case minor groove binding cannot occur. Substitution of guanosines for inosines induces a reversal of the reduced dichroism from negative to positive. Therefore, as anticipated it is the 2-amino group of guanines protruding in this groove which prevents Hoechst 33258 from getting access to the minor groove of GC sequences. The ELD data obtained with the GC-rich biopolymers are consistent with an intercalative binding. Competition experiments performed with the intercalating drug proflavine lend credence to the involvement of an intercalative binding rather than to an external or major groove binding of Hoechst 33258 at GC sequences.     

Phosphorescence and optically detected magnetic resonance studies of echinomycin-DNA complexes

Echinomycin complexes with polymeric DNAs and model duplex oligonucleotides have been studied by low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoxaline chromophores of the drug used as intrinsic probes. Although not optically resolved, plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of echinomycin, which was ascribed to the presence of two distinct quinoxaline triplet-state environments (referred to as the blue and red triplet states of echinomycin in this report). We think that a likely origin of the two triplet states of echinomycin is the occurrence of two or more distinct conformations of the drug in aqueous solutions. Spectroscopically observed perturbations of the triplet-state properties of echinomycin such as the phosphorescence emission spectrum, phosphorescence lifetime, ODMR spectrum, and zero-field splitting (zfs) energies were investigated upon drug binding to the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2]], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, and calf thymus. Echinomycin bisintercalation complexes with the self-complementary oligonucleotides d(ACGT), d(CGTACG), and d(ACGTACGT), which are thought to model drug binding sites, were also investigated. Phosphorescence and ODMR spectroscopic results indicate that the quinoxaline chromophores of the drug are involved in aromatic stacking interactions in complexes with the natural DNAs as evidenced by red shifts in the phosphorescence 0,0 band of the drug, a small but significant reduction in the phosphorescence lifetime of the red triplet state, and reduction of the zfs D-value of both the blue and red triplet states upon drug complexation. These changes in the triplet-state properties of echinomycin are consistent with stacking interactions that increase the polarizability of the quinoxaline environment. The extent of the reduction of the D parameter for the red triplet state upon complexation with the polymeric DNAs was found to correlate with the binding affinities measured for these targets [Wakelin, L. P. G., & Waring, M. J. (1976) Biochem. J. 157, 721-740], with the single exception of the drug-poly[d(G-C)2] complex, for which an increase in the D-value was noted. In addition, upon drug binding to the natural DNAs, there is a reversal of signal polarity in the ODMR spectra of the red triplet state. Among the synthetic DNA polymers investigated, a reversal of ODMR signal polarity was found only with the echinomycin-poly[d(A-T)2] complex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of isoleucine upon quinomycin biosynthesis by Streptomyces sp. 732

When dl-isoleucine was added to an ammonium nitrate-maltose medium during cultivation of Streptomyces sp. 732, quinomycin B formation was selectively enhanced (from 3 to 70% of the quinomycin mixture) and quinomycin C synthesis was inhibited completely. In addition, two new quinomycins, designated quinomycins D and E, were produced in the presence of isoleucine. These compounds were found to contain N-methylalloisoleucine. In experiments with isoleucine enantiomorphs, it was determined that the order of effectiveness for quinomycin B synthesis was dl-isoleucine > d-isoleucine > l-isoleucine. The extent to which quinomycin B synthesis is enhanced depends upon the concentration and the time of addition of isoleucine to the medium. The effect of dl-isoleucine was reduced to some extent by the addition of l-valine. It is conceivable that amino acids which are precursors of the N-methylamino acids in quinomycin can regulate quinomycin formation.     

Synthesis and DNA binding studies of a new asymmetric cyanine dye binding in the minor groove of [poly(dA-dT)]2

A new asymmetric cyanine dye has been synthesised and its interaction with different DNA has been investigated. In this dye, BEBO, the structure of the known intercalating cyanine dye BO has been extended with a benzothiazole substituent. The resulting crescent-shape of the molecule is similar to that of the well-known minor groove binder Hoechst 33258. Indeed, comparative studies of BO illustrate a considerable change in binding mode induced by this structural modification. Linear and circular dichroism studies indicate that BEBO binds in the minor groove to [poly (dA-dT)](2), but that the binding to calf thymus DNA is heterogeneous, although still with a significant contribution of minor groove binding. Similar to other DNA binding asymmetric cyanine dyes, BEBO has a large increase in fluorescence intensity upon binding and a relatively large quantum yield when bound. The minor groove binding of BEBO to [poly (dA-dT)](2) affords roughly a 180-fold increase in intensity, which is larger than to that of the commonly used minor groove binding probes DAPI and Hoechst 33258.     

Molecular recognition of DNA by small molecules: AT base pair specific intercalative binding of cytotoxic plant alkaloid palmatine

The base dependent binding of the cytotoxic alkaloid palmatine to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by competition dialysis, spectrophotometric, spectrofluorimetric, thermal melting, circular dichroic, viscometric and isothermal titration calorimetric (ITC) studies. Binding of the alkaloid to various polynucleotides was dependent upon sequences of base pairs. Binding data obtained from absorbance measurements according to neighbour exclusion model indicated that the intrinsic binding constants decreased in the order poly(dA).poly(dT)>poly(dA-dT).poly(dA-dT)>poly(dG-dC).poly(dG-dC)>poly(dG).poly(dC). This affinity was also revealed by the competition dialysis, increase of steady state fluorescence intensity, increase in fluorescence quantum yield, stabilization against thermal denaturation and perturbations in circular dichroic spectrum. Among the polynucleotides, poly(dA).poly(dT) showed positive cooperativity at binding values lower than r=0.05. Viscosity studies revealed that in the strong binding region, the increase of contour length of DNA depended strongly on the sequence of base pairs being higher for AT polymers and induction of unwinding-rewinding process of covalently closed superhelical DNA. Isothermal titration calorimetric data showed a single entropy driven binding event in the AT homo polymer while that with the hetero polymer involved two binding modes, an entropy driven strong binding followed by an enthalpy driven weak binding. These results unequivocally established that the alkaloid palmatine binds strongly to AT homo and hetero polymers by mechanism of intercalation.     

Interaction of isoquinoline alkaloid palmatine with deoxyribonucleic acids: binding heterogeneity, and conformational and thermodynamic aspects

The binding heterogeneity, conformational aspects, and energetics of the interaction of the cytotoxic plant alkaloid palmatine have been studied with various natural and synthetic DNAs. The alkaloid binds to calf thymus and Escherichia coli DNA that have mixed AT and GC sequences in almost equal proportions with positive cooperativity, while, with Clostridium perfringens and Micrococcus lysodeikticus DNA with predominantly high AT and GC sequences, respectively, noncooperative binding was observed. On further investigation with synthetic DNAs, the binding was observed to be cooperative with polymers like poly(dA).poly(dT) and poly(dG).poly(dC) having poly(purine)poly(pyrimidine) sequences, while with polymers poly(dA-dT).poly(dA-dT), poly(dA-dC).poly(dG-dT) and poly(dG-dC).poly(dG-dC), which have alternating purine-pyrimidine sequences, a non-cooperative binding phenomenon was observed. This suggests the binding heterogeneity of palmatine to the two types of sequences of base pairs. Circular dichroism (CD) studies revealed that the binding induced conformational changes in all the DNAs, but more importantly, the bound alkaloid molecules acquired induced optical activity, and the extent was dependent on the AT content and showed AT base-pair specificity. Energetics of the interaction of the alkaloid studied by highly sensitive isothermal titration calorimetry revealed that the binding was in most cases exothermic and favored by both enthalpy and entropy changes, while, in the case of the homo and hetero AT polymers, the same was predominantly entropy-driven. This study defines base-pair-dependent heterogeneity, conformational aspects, and energetics of palmatine binding to DNA.     

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.     

Conformation of the synthetic DNA poly(amino2dA-dT) duplex in high-salt and aqueous alcohol solutions.

It has previously been demonstrated by other workers that the duplex of a synthetic DNA poly(amino2dA-dT) undergoes a salt-induced conformational isomerization. We show in the present work using circular dichroism that the same isomerization is induced in poly(amino2dA-dT) by various alcohols. The isomerization was originally identified as the B-to-Z and then B-to-A conformational transition of DNA but we demonstrate that the high-salt or alcohol conformation of poly (amino2dA-dT) is the non Z-DNA zig-zag double helix we have previously observed with poly(dA-dT) and called X-DNA. X-DNA is a cesium cation specific conformation of poly(dA-dT) while no similar cation specificity is observed with poly(amino2dA-dT). Thus it appears that the extra amino group attached to A and cesium cations make the same thing; they probably dehydrate the double helix minor groove and relieve its conformational variability. Poly(amino2dA-dT) is exceptionally stable in X-DNA and conditions inducing it are mild, which opens the door to assess its molecular structure.     

Interaction of the nonintercalative antitumour drugs SN-6999 and SN-18071 with DNA: influence of ligand structure on the binding specificity

Binding to DNA's of the non-intercalative ligands SN-6999 and SN-18071 has been studied by means of circular dichroism, UV absorption, thermal melting and for SN-6999 by viscosity measurements. Both antitumour drugs show a preference for dA.dT rich DNA's, but the base pair selectivity of SN-18071 is lower as indicated by some affinity to dG.dC containing duplex DNA. The dA.dT base pair specificity of SN-6999 is comparable to that of netropsin. It forms very stable complexes with dA.dT containing duplex DNA and competes with netropsin binding on DNA. The ligands SN-18071 and pentamidine are totally released from their complexes with poly(dA-dT).poly(dA-dT) by competitive netropsin binding. The results demonstrate that hydrogen bonding capacity of the ligand in addition to other factors strongly contribute to the base sequence specificity in the recognition process of the ligand with DNA. A binding model of SN-6999 with five dA.dT pairs in the minor groove of B-DNA is suggested.     

Triostin A derived hybrid for simultaneous DNA binding and metal coordination

The natural product triostin A is known as an antibiotic based on specific DNA recognition. Structurally, a bicyclic depsipeptide backbone provides a well-defined scaffold preorganizing the recognition motifs for bisintercalation. Replacing the intercalating quinoxaline moieties of triostin A by nucleobases results in a potential major groove binder. The functionalization of this DNA binding triostin A analog with a metal binding ligand system is reported, thereby generating a hybrid molecule with DNA binding and metal coordinating capability. Transition metal ions can be placed in close proximity to dsDNA by means of non-covalent interactions. The synthesis of the nucleobase-modified triostin A analog is described containing a propargylglycine for later attachment of the ligand by click-chemistry. As ligand, two [1,4,7]triazacyclononane rings were bridged by a phenol. Formation of the proposed binuclear zinc complex was confirmed for the ligand and the triostin A analog/ligand construct by high-resolution mass spectrometry. The complex as well as the respective hybrid led to stabilization of dsDNA, thus implying that metal complexation and DNA binding are independent processes.     

DNA binding and uptake by nuclei isolated from plant protoplasts: factors affecting DNA binding and uptake

DNA binding and uptake by nuclei isolated from soybean (Glycine max L. Merr.) protoplasts were investigated using radioactive homogeneous DNA prepared from soybean cells. DNA binding to nuclei was found to decrease drastically with increased incubation time. Total uptake and acid-precipitable uptake reached a maximum after 20 minutes of incubation. Optimum DNA binding and uptake occurred at pH 6 and the process was enhanced by increasing the incubation temperature to 40 C. Salmonella typhimurium DNA and poly ([dA-dT]-[dA-dT]) competitively inhibited DNA binding whereas calf thymus DNA was less competitive; however, Micrococcus lysodeikticus DNA stimulated DNA binding and tobacco mosaic virus RNA had no effect. DNA binding and uptake was enhanced by addition of Mg ions, Ca ions, poly-l-lysine, and ATP. Increasing amounts of EDTA appeared to decrease DNA binding. Pronase strongly inhibited DNA binding and uptake.     

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity,Conformational, Thermodynamic and Cytotoxic Aspects

Background

Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.

Methodology/Principal Findings

Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the in vitro chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.

Conclusions/Significance

Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.