DNA-drug interactions. The crystal structure of d(CGATCG) complexed with daunomycin

The structure of a d(CGATCG)-daunomycin complex has been determined by single crystal X-ray diffraction techniques. Refinement, with the location of 40 solvent molecules, using data up to 1.5 A, converged with a final crystallographic residual, R = 0.25 (RW = 0.22). The tetragonal crystals are in space group P4(1)2(1)2, with cell dimensions of a = 27.98 A and c = 52.87 A. The self-complementary d(CGATCG) forms a distorted right-handed helix with a daunomycin molecule intercalated at each d(CpG) step. The daunomycin aglycon chromophore is oriented at right-angles to the long axis of the DNA base-pairs. This head-on intercalation is stabilized by direct hydrogen bonds and indirectly via solvent-mediated, hydrogen-bonding interactions between the chromophore and its intercalation site base-pairs. The cyclohexene ring and amino sugar substituent lie in the minor groove. The amino sugar N-3' forms a hydrogen bond with O-2 of the next neighbouring thymine. This electrostatic interaction helps position the sugar in a way that results in extensive van der Waals contacts between the drug and the DNA. There is no interaction between daunosamine and the DNA sugar-phosphate backbone. We present full experimental details and all relevant conformational parameters, and use the comparison with a d(CGTACG)-daunomycin complex to rationalize some neighbouring sequence effects involved in daunomycin binding.     

Daunomycin intercalation stabilizes distinct backbone conformations of DNA

Daunomycin is a widely used antibiotic of the anthracycline family. In the present study we reveal the structural properties and important intercalator-DNA interactions by means of molecular dynamics. As most of the X-ray structures of DNA-daunomycin intercalated complexes are short hexamers or octamers of DNA with two drug molecules per doublehelix we calculated a self complementary 14-mer oligodeoxyribonucleotide duplex d(CGCGCGATCGCGCG)2 in the B-form with two putative intercalation sites at the 5'-CGA-3' step on both strands. Consequently we are able to look at the structure of a 1:1 complex and exclude crystal packing effects normally encountered in most of the X-ray crystallographic studies conducted so far. We performed different 10 to 20 ns long molecular dynamics simulations of the uncomplexed DNA structure, the DNA-daunomycin complex and a 1:2 complex of DNA-daunomycin where the two intercalator molecules are stacked into the two opposing 5'-CGA-3' steps. Thereby--in contrast to X-ray structures--a comparison of a complex of only one with a complex of two intercalators per doublehelix is possible. The chromophore of daunomycin is intercalated between the 5'-CG-3' bases while the daunosamine sugar moiety is placed in the minor groove. We observe a flexibility of the dihedral angle at the glycosidic bond, leading to three different positions of the ammonium group responsible for important contacts in the minor groove. Furthermore a distinct pattern of BI and BII around the intercalation site is induced and stabilized. This indicates a transfer of changes in the DNA geometry caused by intercalation to the DNA backbone.     

Antitumor drug nogalamycin binds DNA in both grooves simultaneously: molecular structure of nogalamycin-DNA complex

The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, d[CGT(pS)ACG] and d[m5CGT(pS)Am5CG], were determined at high resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.     

Binding of the antitumor drug nogalamycin and its derivatives to DNA: structural comparison

The three-dimensional molecular structures of the complexes between a novel antitumor drug nogalamycin and its derivative U-58872 with a modified DNA hexamer d[m5CGT(pS)Am5CG] have been determined at 1.7- and 1.8-A resolution, respectively, by X-ray diffraction analyses. Both structures (in space group P6(1)) have been refined with constrained refinement procedure to final R factors of 0.208 (3386 reflections) and 0.196 (2143 reflections). In both complexes, two nogalamycins bind to the DNA hexamer double helix in a 2:1 ratio with the elongated aglycon chromophore intercalated between the CpG steps at both ends of the helix. The aglycon chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. Most of the sugars remain in the C2'-endo pucker family, except three deoxycytidine residues (terminal C1, C7, and internal C5). All nucleotides are in the anti conformation. Specific hydrogen bonds are found in the complex between the drug and guanine-cytosine bases in both grooves of the helix. One hydroxyl group of the aminoglucose donates a hydrogen bond to the N7 of guanine, while the other receives a hydrogen bond from the N4 amino group of cytosine. The orientation of these two hydrogen bonds suggests that nogalamycin prefers a GC base pair with its aglycon chromophore intercalating at the 5'-side of a guanine (between NpG), or at the 3'-side of a cytosine (between CpN) with the sugars pointing toward the GC base pair. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through, suggesting that nogalamycin prefers GC sequences embedded in a stretch of AT sequences.     

Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexed to d(CpGpTpApCpG) at 1.2-A resolution

The crystal structure of a daunomycin-d(CGTACG) complex has been solved by X-ray diffraction analysis and refined to a final R factor of 0.175 at 1.2-A resolution. The crystals are in a tetragonal crystal system with space group P4(1)2(1)2 and cell dimensions of a = b = 27.86 A and c = 52.72 A. The self-complementary DNA forms a six base pair right-handed double helix with two daunomycin molecules intercalated in the d(CpG) sequences at either end of the helix. Daunomycin in the complex has a conformation different from that of daunomycin alone. The daunomycin aglycon chromophore is oriented at right angles to the long dimension of the DNA base pairs, and the cyclohexene ring A rests in the minor groove of the double helix. Substituents on this ring have hydrogen-bonding interactions to the base pairs above and below the intercalation site. O9 hydroxyl group of the daunomycin forms two hydrogen bonds with N3 and N2 of an adjacent guanine base. Two bridging water molecules between the drug and DNA stabilize the complex in the minor groove. In the major groove, a hydrated sodium ion is coordinated to N7 of the terminal guanine and the O4 and O5 of daunomycin with a distorted octahedral geometry. The amino sugar lies in the minor groove without bonding to the DNA. The DNA double helix is distorted with an asymmetrical rearrangement of the backbone conformation surrounding the intercalator drug. The sugar puckers are C1,C2'-endo, G2,C1'-endo, C11,C1'-endo, and G12,C3'-exo. Only the C1 residue has a normal anti-glycosyl torsion angle (chi = -154 degrees), while the other three residues are all in the high anti range (average chi = -86 degrees). This structure allows us to identify three principal functional components of anthracycline antibiotics: the intercalator (rings B-D), the anchoring functions associated with ring A, and the amino sugar. The structure-function relationships of daunomycin binding to DNA as well as other related anticancer drugs are discussed.     

The interaction of adriamycin and adriamycin analogues with nucleic acids in the B and A conformations.

The reinforced intercalative binding to DNA typical of adriamycin and daunomycin can still occur if there is epimerisation at C4' or if the O-methyl group is lost or if the 9-substituents are deleted or if the 4'-hydroxyl group is lost. In the latter two cases however, there is a reduction in affinity for the DNA, supporting the suggested role of the 9-hydroxyl and 4'-hydroxyl groups in secondary stabilization of the complex. Epimerisation at C-1' or at C-3' alters but does not abolish the intercalative mode of binding to DNA whereas epimerisation at C-7 precludes intercalation of the chromophore into the helix of DNA. In contrast to the interaction with the B-form found in DNA, the parent drugs do not intercalate into nucleic acids possessing the A-conformation and none of the above-mentioned structural changes will allow intercalation into A-form nucleic acids.     

NMR studies of the interaction of the antibiotic nogalamycin with the hexadeoxyribonucleotide duplex d(5'-GCATGC)2

1H resonance assignments in the NMR spectra of the self-complementary hexadeoxyribonucleoside pentaphosphate d(5'-GCATGC)2 and its complex with the antibiotic nogalamycin, together with interproton distance constraints obtained from two-dimensional nuclear Overhauser effect (NOE) spectra, have enabled us to characterize the three-dimensional structure of these species in solution. In the complex described, two drug molecules are bound per duplex, in each of two equivalent binding sites, with full retention of the dyad symmetry. Twenty-eight NOE distance constraints between antibiotic and nucleotide protons define the position and orientation of the bound drug molecule. Nogalamycin intercalates at the 5'-CA and 5'-TG steps with the major axis of the anthracycline chromophore aligned approximately at right angles to the major axes of the base pairs. The nogalose sugar occupies the minor groove of the helix and makes many contacts with the deoxyribose moieties of three nucleotides along one strand of the duplex in the 5'-TGC segment. The charged dimethylamino group and hydroxyl functions of the bicyclic sugar lie in the major groove juxtaposed to the guanine base, the bridging atoms of the bicyclic sugar making contacts with the methyl group of the thymine. Thus the antibiotic is not symmetrically disposed in the intercalation site but is in close contact in both grooves with atoms comprising the 5'-TGC strand. The intercalation cavity is wedge-shaped, the major axes of the base pairs forming the site being tilted with respect to one another. All base-pair hydrogen-bonding interactions are maintained in the complex, and there is no evidence for Hoogsteen pairing. The free duplex adopts a regular right-handed B-type conformation in which all glycosidic bond angles are anti and all sugar puckers lie in the C2'-endo range. In the complex the glycosidic bond angles and the sugar puckers deviate little from those observed for the duplex alone. The presence of two bound nogalamycin molecules substantially slows the "breathing" motions of the base pairs forming the intercalation cavity, and the observation of two downfield-shifted resonances in the 31P NMR spectrum of the complex suggests a pronounced local helix unwinding at the drug binding site. The footprinting data of Fox and Waring [Fox, K.R., & Waring, M.J. (1986) Biochemistry 25, 4349-4356] imply that the highest affinity binding sites of nogalamycin have the sequence 5'-GCA (or 5'-TGC).(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA-drug interactions. The crystal structures of d(TGTACA) and d(TGATCA) complexed with daunomycin

The anticancer drug daunomycin has been co-crystallized with the hexanucleotide duplex sequences d(TGTACA) and d(TGATCA) and single crystal X-ray diffraction studies of these two complexes have been carried out. Structure solution of the d(TGTACA) and d(TGATCA) complexes to 1.6 and 1.7 Angstrom resolution, respectively, shows two daunomycin molecules bound to the DNA hexamer. Binding occurs via intercalation of the drug chromophore at the d(TpG) step, and hydrogen bonding interactions involving the drug, DNA and solvent molecules. The daunomycin sugar is located in the minor groove of the DNA hexamer and is stabilized by hydrogen bonds between the amino group of the sugar and functional groups on the floor of the groove. The amino sugar of the d(TGATCA) duplex interacts directly with the DNA sequence, while in the d(TGTACA) duplex, the interaction is via solvent molecules. Two other complexes d(CGTACG)-daunomycin and d(CGATCG)-daunomycin have previously been structurally characterized. Comparison of the four structures with daunomycin bound to the triplet sequences 5'TGT, 5'TGA, 5'CGT and 5'CGA reveals changes in the conformation of both the DNA hexamer and the daunomycin upon complexation, as well as the hydrogen bonding and van der Waals' interactions.     

Electrostatic factors in DNA intercalation

The factors that determine the binding of a chromophore between the base pairs in DNA intercalation complexes are dissected. The electrostatic potential in the intercalation plane is calculated using an accurate ab initio based distributed multipole electrostatic model for a range of intercalation sites, involving different sequences of base pairs and relative twist angles. There will be a significant electrostatic contribution to the binding energy for chromophores with a predominantly positive electrostatic potential, but this varies significantly with sequence, and somewhat with twist angle. The usefulness of these potential maps for understanding the binding of intercalators is explored by calculating the electrostatic binding energy for 9-aminoacridine, ethidium, and daunomycin in a variety of model binding sites. The electrostatic forces play a major role in the positioning of an intercalating 9-aminoacridine and a significant stabilizing role in the binding of ethidium in its sterically constrained position, but the intercalation of daunomycin is determined by the side-chain binding. Sequence preferences are likely to be determined by a complex and subtle mixture of effects, with electrostatics being just one component. The electrostatic binding energy is also unlikely to be a major determinant of the twist angle, as its variation with angle is modest for most intercalation sites. Overall, the electrostatic potential maps give guidance on how positively charged chromophores can be chemically adapted by heteroatomic substitution to optimise their 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.     

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 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 sinigle exponential. Dissociation of daunomycin, adrianmycin 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 antibotics 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.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

NMR studies on the binding of antitumor drug nogalamycin to DNA hexamer d(CGTACG).

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.     

Solution structure of the nogalamycin-DNA complex

The nogalamycin-d(A-G-C-A-T-G-C-T) complex (two drugs per duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Two equivalents of nogalamycin binds to the self-complementary octanucleotide duplex with retention of 2-fold symmetry in solution. We have assigned the proton resonances of nogalamycin and the d(A1-G2-C3-A4-T5-G6-C7-T8) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site on duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the nogalamycin and the DNA in the complex. The molecular dynamics calculations were guided by 274 intramolecular nucleic acid distance constraints, 90 intramolecular nogalamycin distance constraints, and 104 intermolecular distance constraints between nogalamycin and the nucleic acid protons in the complex. The aglycon chromophore intercalates at (C-A).(T-G) steps with the long axis of the aglycon approximately perpendicular to the long axis of the flanking C3.G6 and A4.T5 base pairs. The aglycon selectively stacks over T5 and G6 on the T5-G6-containing strand with the aglycon edge containing OH-4 and OH-6 substituents directed toward the C3-A4-containing strand. The C3.G6 and A4.T5 base pairs are intact but buckled at the intercalation site with a wedge-shaped alignment of C3 and A4 on the C3-A4 strand compared to the parallel alignment of T5 and G6 on the T5-G6 strand in the complex. The nogalose sugar in a chair conformation, the aglycon ring A in a half-chair conformation, and the COOCH3-10 side chain form a continuous domain that is sandwiched within the walls of the minor groove and spans the three base pair (G2-C3-A4).(T5-G6-C7) segment. The nogalose ring is positioned in the minor groove such that its nonpolar face is directed toward the G6-C7 sugar-phosphate backbone while its polar face containing OCH3 groups is directed toward the G2-C3 sugar-phosphate backbone in the complex. The intermolecular contacts include a nonpolar patch of aglycon (CH3-9) and nogalose (CH3-3') methyl groups forming van der Waals contacts with the base-sugar residues in the minor groove and intermolecular hydrogen bonds involving the amino groups of G2 and G6 with the ether oxygens OCH3-3' and O7, respectively, on the nogalose sugar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character.     

X-ray diffraction and infrared circular dichroism of DNA-violamycin B1 complexes

X-ray diffraction and infrared linear dichroism of oriented samples of DNA-violamycin B1 complexes have been studied at different antibiotic/DNA phosphate ratios (r) as a function of relative humidity. Violamycin B1 binds to DNA according to the intercalation as well as to the outside binding model. At low r values, where the intercalation predominates the unwinding angle of DNA helix is between 6 degrees and 12 degrees per intercalation site as followed from the dependence of the pitch of helix versus r. At r greater than or equal to 0.17 the intercalation sites are saturated and the outside binding becomes prevalent; however the violamycin B1 chromophore is still oriented in the plane of DNA bases. Conformational mobility of DNA in the violamycin B1 complexes is largely inhibited compared with pure DNA, but it is higher than that of the daunomycin complexes. At least 30% of DNA in violamycin complexes has A conformation at the medium humidities as followed by IR linear dichroism. In the case of x-ray diffraction the A conformation was not detected. The distance between DNA molecules in the complex is found to be 23.2 A, that is 2 A less than in pure DNA at the same conditions and it does not depend upon r.     

Energetics and stereochemistry of DNA complexation with the antitumor AT specific intercalators tilorone and m-AMSA.

Computations by the SIBFA method on the intercalative interaction energies of tilorone and m-AMSA with B-DNA representative oligonucleotides account for the specificity of these antitumor drugs for AT sites and minor groove intercalation. In tilorone this specificity is due to the strong preference of the side chains for the minor groove, which overcomes the preference of the chromophore for a GC intercalation site. In m-AMSA the specificity is due to the combined preference of both the chromophore and the anilino side chain for AT intercalation site and minor groove, respectively. o-AMSA is shown to manifest a similar (although significantly less pronounced specificity) as m-AMSA but a higher affinity for DNA. A comparison of the energetics and stereochemistry of intercalative binding to DNA of m-AMSA (AT minor groove specific) and 9-aminoacridine-4-carboxamide (GC major groove specific), which possess the same chromophore and differ only by the nature and position of the side chains, shows the possibility of important variations in the intercalative behaviour of chromophoric drugs as a function of the substituent groups attached to them.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.     

1H-NMR study of the interaction of daunomycin with B-DNA helices of methylated oligodeoxynucleotides.

The interaction of daunomycin with B-DNA double helices of several methylated deoxynucleotides, d(C-G-m5C-G), d(m5C-G-C-G), d(C-G-m5C-G-C-G) and d(m5C-G-C-G-m5C-G) in solution was investigated by 1H-NMR spectroscopy at 500 MHz. At low temperature (t less than 20 degrees C for the tetramer and t less than 40 degrees C for the hexamers), several daunomycin-DNA complexes were observed in slow exchange with the drug-free DNA duplexes. The presence of daunomycin in a self-complementary double helix cancels the conformational symmetry of the two strands; the proton signals can split into several others owing to the difference between free and intercalated duplexes and to the many possible intercalation sites in a duplex (three for a tetramer, five for an hexamer). A model relating the chemical shifts of splitted proton signals to the various intercalated duplex conformations was given. The results show that one daunomycin molecule is associated with one duplex and that it can enter any intercalation site with equal probability; no side-effects were observed even for very short helices (of a tetramer). In the case of d(C-G-m5C-G) the association constant and the dissociation and association rates of the intercalated complex were evaluated.     

Anthracycline antibiotic arugomycin binds in both grooves of the DNA helix simultaneously: an NMR and molecular modelling study.

Perturbations to the 1H and 31P chemical shifts of DNA resonances together with twenty-four intermolecular nuclear Overhauser effects show that the anthracycline antibiotic arugomycin intercalates between the basepairs of the hexamer duplex d(5'-GCATGC)2 at the 5'-CpA and 5'-TpG binding sites. In the complex two drug molecules are bound per duplex with full retention of the dyad symmetry. Arugomycin adopts a threaded binding orientation with chains of sugars positioned in both the major and minor groove of the helix simultaneously. The complex is stabilized by hydrogen bonding, electrostatic and van der Waals interactions principally in the major groove and involving substituents on the rigidly oriented bicycloamino-glucose sugar of the antibiotic. A specific hydrogen bond is identified between the C2'-hydroxyl and the guanine N7 at the intercalation site. Together, interactions in the major groove appear to account for the intercalation specificity of arugomycin that requires both a guanine and thymine at the intercalation site. We are unable to identify any sequence specific interactions between the minor groove and the arugarose sugar (S1) which binds only weakly, through van der Walls contacts, over the d(GCA).d(TGC) trinucleotide sequence. The data indicate that the sugar chains of arugomycin are flexible and play little part in the interaction of the antibiotic with DNA. The intensity of sequential internucleotide NOEs identifies the intercalation site as being assymmetric. A family of conformers computed using restrained energy minimisation and molecular dynamics indicate that basepair buckling is a feature of the anthracycline intercalation site that may serve to maximise intermolecular van der Waals interactions by wrapping the basepairs around the antibiotic chromophore.