NMR investigation of mithramycin A binding to d(ATGCAT)2: a comparative study with chromomycin A3

The binding of mithramycin A to the d(A1T2G3C4A5T6) duplex was investigated by 1H NMR and found to be similar to that of its analogue chromomycin A3. In the presence of Mg2+, mithramycin binds strongly to d(ATGCAT)2. On the basis of the two-dimensional NOESY spectrum, the complex formed possesses C2 symmetry at a stoichiometry of two drugs per duplex (2:1) and is in slow chemical exchange on the NMR time scale. NOESY experiments reveal contacts from the E-pyranose of mithramycin to the terminal and nonterminal adenine H2 proton of DNA and from the drug hydroxyl proton to both G3NH2 protons, C4H1' proton, and A5H1' proton. These data place the drug chromophore and E pyranose on the minor groove side of d(ATGCAT)2. NOE contacts from the A-, B-, C-, and D-pyranoses of mithramycin to several deoxyribose protons suggest that the A- and B-rings are oriented along the sugar-phosphate backbone of G3-C4, while the C- and D-rings are located along the sugar-phosphate backbone of A5-T6. These drug-DNA contacts are very similar to those found for chromomycin binding to d(ATGCAT)2. Unlike chromomycin, the NOESY spectrum of mithramycin at the molar ratio of one drug per duplex reveals several chemical exchange cross-peaks corresponding to the drug-free and drug-bound proton resonances. From the intensity of these cross-peaks and the corresponding diagonal peaks, the off-rate constant was estimated to be 0.4 s-1. These data suggest that the exchange rate of mithramycin binding to d(ATGCAT)2 is faster than that of chromomycin.     

H-NMR studies on d(GCTTAAGC)2 and its complex with berenil.

Two-dimensional (2D) 1H-NMR spectroscopy has been used to analyze the structure of d(GCTTAAGC)2 and its interaction with berenil in solution. Nuclear Overhauser enhancement connectivities enabled sequential assignments of nearly all proton resonances in the self-complementary octamer duplex and demonstrated that the oligonucleotide is primarily in a B-type conformation. No major conformational changes were observed by the addition of berenil, but proton resonances of the two adenosine nucleotides shifted substantially. Intermolecular nuclear Overhauser effects between berenil and the DNA duplex revealed that the drug binds via the minor groove of d(GCTTAAGC)2 in the A.T-base-pair region. At 18 degrees C the twofold symmetry of the duplex is preserved on berenil binding. However, strongly shifted proton resonances broadened significantly. A model is proposed for the berenil-d(GCTTAAGC)2 complex involving fast exchange of berenil between two equivalent symmetry-related binding sites, which span the 5'-TAA-3' region and are asymmetrically disposed with respect to the dyad axis of the duplex. These results are compared with previous studies on the berenil-d(GCAATTGC)2 complex.     

The three-dimensional structure of the 4:1 mithramycin:d(ACCCGGGT)(2) complex: evidence for an interaction between the E saccharides

Mithramycin and chromomycin, two antitumor drugs, each having an identical aglycone and nearly identical disaccharide and trisaccharide side chains, have differing binding properties to a small oligonucleotide, d(ACCCGGGT)(2) (M. A. Keniry et al., Journal of Molecular Biology, 1993, Vol. 231, pp. 753-767). In order to understand the forces that induce four mithramycin molecules to bind to d(ACCCGGGT)(2) instead of two drug molecules in the case of chromomycin, the structure of the 4:2:1 mithramycin: Mg(2+):d(ACCCGGGT)(2) complex was investigated by (1)H-nmr and restrained molecular dynamics. The resulting three-dimensional model showed that in order to accommodate the close approach of one neighboring mithramycin dimer, the inwardly directed CDE saccharide chain of the neighboring mithramycin dimer undergoes a conformational change such that the E saccharide no longer spans the minor groove but reorients so that the hydrophilic face of the E saccharides from the two dimers oppose each other. Two hydrogen bonds are formed between the hydroxyl groups of the two opposing E saccharide groups. The results are interpreted in terms of the differences in stereochemistry and functional group substitutions between mithramycin and chromomycin. A mithramycin dimer is able to self-associate on an oligonucleotide template because it has two hydroxyl groups on the same face of its terminal E saccharide. A chromomycin dimer is unable to self-associate because one of these hydroxyl groups is acetylated and the neighboring hydroxyl group has a stereochemistry that cannot permit close contact of the hydroxyl group with a neighbouring chromomycin dimer.Copyright 2000 John Wiley & Sons, Inc.     

NMR studies of the interaction of chromomycin A3 with small DNA duplexes. Binding to GC-containing sequences

The interaction of chromomycin A3 with the oligodeoxyribonucleotides 1, d(ATGCAT), 2, d(ATCGAT), 3, d(TATGCATA), and 4, d(ATAGCTAT), has been investigated by 1H and 31P NMR. In the presence of Mg2+, chromomycin binds strongly to the three GC-containing oligomers 1, 3, and 4 but not to the CG-containing oligomer 2. The proton chemical shift changes for 1 and 3 are similar, and these DNA duplexes appear to bind with a stoichiometry of 2 drugs:1 Mg2+:1 duplex. The same stoichiometry of 2 drugs:1 duplex is confirmed with 4; however, proton chemical shift changes differ. An overall C2 symmetry is exhibited by the drug complex with 1, 3, and 4. At a molar ratio of 2.0 (drugs:duplex), no free DNA proton NMR signals remain. Two-dimensional nuclear Overhauser exchange spectroscopy (NOESY) of the saturated chromomycin complex with 1 and 3 positions both chromomycinone hydroxyls and the E carbohydrates in the minor groove and provides evidence suggesting that the B carbohydrates lie on the major-groove side. This is supported by several dipolar coupling cross-peaks between the drug and the DNA duplex. Drug-induced conformational changes in duplex 1 are evaluated over a range of NOESY mixing times and found to possess some characteristics of both B-DNA and A-DNA, where the minor groove is wider and shallower. A widening of the minor groove is essential for the DNA duplex to accommodate two drug molecules. This current minor-groove model is a substantial revision of our earlier major-groove model [Keniry, M.A., Brown, S.C., Berman, E., & Shafer, R.H. (1987) Biochemistry 26, 1058-1067] and is in agreement with the model recently proposed by Gao and Patel [Gao, X., & Patel, D. J. (1989a) Biochemistry 28, 751-762].     

Anthracycline binding to DNA. High-resolution structure of d(TGTACA) complexed with 4'-epiadriamycin.

Crystallographic methods have been applied to determine the high-resolution structure of the complex formed between the self-complementary oligonucleotide d(TGTACA) and the anthracycline antibiotic 4'-epiadriamycin. The complex crystallises in the tetragonal system, space group P4(1)2(1)2 with a = 2.802 nm and c = 5.293 nm, and an asymmetric unit consisting of a single DNA strand, one drug molecule and 34 solvent molecules. The refinement converged with an R factor of 0.17 for the 2381 reflections with F greater than or equal to 3 sigma F in the resolution range 0.70-0.14 nm. Two asymmetric units associate such that a distorted B-DNA-type hexanucleotide duplex is formed incorporating two drug molecules that are intercalated at the TpG steps. The amino sugar of 4'-epiadriamycin binds in the minor groove of the duplex and displays different interactions from those observed in previously determined structures. Interactions between the hydrophilic groups of the amino sugar and the oligonucleotide are all mediated by solvent molecules. Ultraviolet melting measurements and comparison with other anthracycline-DNA complexes suggest that these indirect interactions have a powerful stabilising effect on the complex.     

An NMR study of the covalent and noncovalent interactions of CC-1065 and DNA

The binding of the antitumor drug CC-1065 has been studied with nuclear magnetic resonance (NMR) spectroscopy. This study involves two parts, the elucidation of the covalent binding site of the drug to DNA and a detailed investigation of the noncovalent interactions of CC-1065 with a DNA fragment through analysis of 2D NOE (NOESY) experiments. A CC-1065-DNA adduct was prepared, and an adenine adduct was released upon heating. NMR (1H and 13C) analysis of the adduct shows that the drug binds to N3 of adenine by reaction of its cyclopropyl group. The reaction pathway and product formed were determined by analysis of the 13C DEPT spectra. An octamer duplex, d(CGATTAGC.GCTAATCG), was synthesized and used in the interaction study of CC-1065 and the oligomer. The duplex and the drug-octamer complex were both analyzed by 2D spectroscopy (COSY, NOESY). The relative intensity of the NOEs observed between the drug (CC-1065) and the octamer duplex shows conclusively that the drug is located in the minor groove, covalently attached to N3 of adenine 6 and positioned from the 3'----5' end in relation to strand A [d(CGATTA6GC)]. A mechanism for drug binding and stabilization can be inferred from the NOE data and model-building studies.     

Structure and dynamics of distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at low drug:DNA ratios

Two-dimensional NMR has been used to study the interaction of distamycin A with d(CGCAAATTGGC):d(GCCAATTTGCG) at low and intermediate drug:DNA ratios (less than 2.0). Drug-DNA contacts were identified by nuclear Overhauser effect spectroscopy, which also served to monitor exchange of the drug between different binding sites. At low drug:DNA ratios (0.5), distamycin A binds in two orientations within the five central A-T base pairs and has a preference (2.2:1) for binding with the formyl end directed toward the 5' side of the A-rich strand. The pattern of drug-DNA contacts corresponding to the preferred binding orientation are consistent with the drug sliding between adjacent AAAT and AATT binding sites at a rate that is fast on the NMR time scale. Similarly, the pattern of NOEs associated with the less favored orientation are consistent with the drug sliding between adjacent AATT and ATTT sites, again in fast exchange. Off-rates for the drug from the major and minor binding orientations were measured to be 2.4 +/- 1.5 and 3.3 +/- 1.5 s-1, respectively, at 35 degrees C. At intermediate drug:DNA ratios (1.3) exchange of the drug between the two one-drug and the two sites of a two-drug complex is observed. Off-rates for both drugs from the 2:1 complex were measured to be 1.0 +/- 0.5 s-1 (35 degrees C).     

Solution structure of the luzopeptin-DNA complex.

The luzopeptin-d(C-A-T-G) complex (1 drug/duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. One equivalent of luzopeptin binds to the self-complementary tetranucleotide duplex with the 2-fold symmetry of the antitumor agent and the DNA oligomer retained on complex formation. We have assigned the exchangeable and nonexchangeable proton resonances of luzopeptin and the d(C-A-T-G) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site in duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the luzopeptin and the DNA in the complex. The molecular dynamics calculations were guided by 140 intramolecular nucleic acid distance constraints, 74 intramolecular luzopeptin distance constraints, and 96 intermolecular distance constraints between luzopeptin and the nucleic acid protons in the complex. The quinoline rings of luzopeptin bisintercalate at d(C-A).d(T-G) steps in the d(C-A-T-G) duplex and sandwich two Watson-Crick A.T base pairs between the bisintercalation site. The long axis of the quinoline rings are collinear with the long axis of the flanking Watson-Crick C1.G4 and A2.T3 base pairs such that the OCH3-6 group is directed toward the C1-A2 step and the OH-3 group is directed toward the T3-G4 step in the complex. The quinoline chromophore stacks with purines on both strands, with the quinoline A ring stacked on A2 and the quinoline B ring stacked on G4 in the complex. The C1.G4 and A2.T3 base pairs that flank the intercalation sites are parallel to each other with partial overlap of T3 and G4 in the T3-G4 step but no overlap of C1 and A2 in the C1-A2 step in the complex. The cyclic depsipeptide ring of luzopeptin is positioned in the minor groove of the d(C-A-T-G) duplex with the oligopeptide and oligonucleotide chains running antiparallel to each other. The cyclic depsipeptide backbone of luzopeptin exhibits cis peptide bonds at Pyr-Gly and Gly-Sar steps in the luzopeptin-d(C-A-T-G) complex in solution, in contrast to all trans peptide bonds for free luzopeptin in the crystalline state.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamics and binding mode of Hoechst 33258 to d(GTGGAATTCCAC)2 in the 1:1 solution complex as determined by two-dimensional 1H NMR.

We have investigated the interaction of the bisbenzimidazole derivative Hoechst 33258 with the self-complementary dodecadeoxynucleotide duplex d(GTGGAATTCCAC)2 using one-dimensional (1D) and two-dimensional (2D) proton nuclear magnetic resonance (1H NMR) spectroscopy. To monitor the extent of complex formation, we used the imino proton region of the 1D 1H NMR spectra acquired in H2O solution. These spectra show that the DNA duplex loses its inherent C2v symmetry upon addition of the drug, indicating that the two molecules form a kinetically stable complex on the NMR time scale (the lifetime of the complex has been measured to be around 450 ms). We obtained sequence-specific assignments for all protons of the ligand and most protons of each separate strand of the oligonucleotide duplex using a variety of homonuclear 2D 1H NMR experiments. The aromatic protons of the DNA strands, which are symmetrically related in the free duplex, exhibit exchange cross peaks in the complex. This indicates that the drug binds in two equivalent sites on the 12-mer, with an exchange rate constant of 2.2 +/- 0.2 s-1. Twenty-five intermolecular NOEs were identified, all involving adenine 2 and sugar 1' protons of the DNA and protons in all four residues of the ligand, indicating that Hoechst 33258 is located in the minor groove at the AATT site. Only protons along the same edge of the two benzimidazole moieties of the drug show NOEs to DNA protons at the bottom of the minor groove. Using molecular mechanics, we have generated a unique model of the complex using distance constraints derived from the intermolecular NOEs. We present, however, evidence that the piperazine group may adopt at least two locally different conformations when the drug is bound to this dodecanucleotide.     

Actinomycin D binds strongly to d(CGACGACG) and d(CGTCGTCG)

Earlier calorimetric studies had indicated that despite the absence of a GpC sequence, the self-complementary octamer d(CGTCGACG) binds strongly to actinomycin D (ACTD) with high cooperativity and a 2:1 drug/duplex ratio. A subsequent optical spectral study with related oligomers led us to suggest that ACTD may likely stack at the G. C basepairs of the duplex termini. New findings are reported herein to indicate that despite the lack of complete self-complementarity, oligomers of d(CGXCGXCG) [X = A or T] motif exhibit unusually strong ACTD affinities with binding constants of roughly 2 x 10(7) M(-1) and binding densities of 1 drug molecule per strand. The ACTD binding affinity for the corresponding heteroduplex obtained by annealing these two oligomers is, however, considerably reduced. Although spectroscopic results with related oligomers obtained by removing, replacing, or appending bases at the termini appear to be consistent with the end-stacking model, capillary electrophoretic (CE) evidence provides additional insights into the binding mode. CE experiments with the self-complementary oligomers d(CGAGCTCG) and d(CGTCGACG) revealed contrasting migration patterns in the presence of ACTD, with mobility retardation and acceleration exhibited by the GpC- and non-GpC-containing octamers, respectively, whereas the X/X-mismatched d(CGXCGXCG) experienced retardation. These results, along with those of related oligomers, suggest that ACTD may in fact stack at the duplex stem end of a monomeric hairpin or at the 3'-end of dG as a single strand. The seemingly cooperative ACTD binding and the curved Scatchard plot for the self-complementary d(CGTCGACG) may thus be attributed to the drug-induced duplex denaturation resulting from strong binding to single strands of d(CGXCGYCG) motif. Detailed structural information on the ACTD-DNA complexes, however, must await further NMR investigations.     

Catalytic activities of synthetic octadeoxyribonucleotides as coenzymes of poly(ADP-ribose) polymerase and the identification of a new enzyme inhibitory site

The catalytic activity of highly purified poly(ADP-ribose) polymerase was determined at constant NAD+ concentration and varying concentrations of sDNA or synthetic octadeoxyribonucleotides of differing composition. The coenzymic activities of deoxyribonucleotides were compared in two ways: graphic presentation of the activation of poly(ADP-ribose) polymerase in the presence of a large concentration range of deoxyribonucleotides and by calculating kD values for the deoxyribonucleotides. As determined by method i, auto-mono-ADP-ribosylation of the enzyme protein at 25 nM NAD+ was maximally activated at 1:1 octamer/enzyme molar ratios by the octadeoxyribonucleotide derived from the regulatory region of SV40 DNA (duplex C). At a 0.4:1 sDNA/enzyme ratio, sDNA was the most active coenzyme for mono-ADP-ribosylation. At 200 microM NAD+, resulting in polymer synthesis and with histones as secondary polymer acceptors, duplex C was the most active coenzyme, and the octamer containing the steroid hormone receptor binding consensus sequence of DNA was a close second, whereas sDNA exhibited an anomalous biphasic kinetics. sDNA was effective on mono-ADP-ribosylation at a concentration 150-200 -times lower than on polymer formation. When comparison of deoxyribonucleotides was based on method ii (kD values), by far the most efficiently binding coenzyme for both mono and polymer synthesis was sDNA, followed by duplex C, with (dA-dT)8 exhibiting the weakest binding. The synthetic molecule 6-amino-1,2-benzopyrone (6-aminocoumarin) competitively inhibited the coenzymic function of synthetic octadeoxyribonucleotides at constant concentration of NAD+, identifying a new inhibitory site of poly(ADP-ribose) polymerase.     

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.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

2D NMR investigation of the binding of the anticancer drug actinomycin D to duplexed dATGCGCAT: conformational features of the unique 2:1 adduct

One- and two-dimensional NMR studies on the oligomer dA1T2G3C4G5C6A7T8, with and without actinomycin D (ActD), were conducted. Analysis of the NMR data, particularly 2D NOE intensities, revealed that the free oligonucleotide is a duplex in a standard right-handed B form. At the ratio of 1 ActD/duplex (R = 1), 1D NMR studies indicate that two 1:1 unsymmetric complexes form in unequal proportions with the phenoxazone ring intercalated at a GpC site, in agreement with previous studies [Scott, E.V., Jones, R.L., Banville, D.L., Zon, G., Marzilli, L.G., & Wilson, W.D. (1988) Biochemistry 27, 915-923]. The 2D COSY data also confirm this interpretation since eight cytosine H6 to H5 and two ActD H8 to H7 cross-peaks are observed. At R = 2, both COSY and NOESY spectra confirm the formation of a unique 2:1 species with C2 symmetry. The oligomer remains in a right-handed duplex but undergoes extreme conformational changes both at and adjacent to the binding site. The deoxyribose conformation of T2, C4, and C6 shifts from primarily C2'-endo in the free duplex to an increased amount of C3'-endo in the 2:1 complex as revealed by the greater intensity of the base H6 to 3' NOE cross-peak relative to the intensity of the H6 to H2' NOE cross-peak. This conformational change widens the minor groove and should help alleviate the steric crowding of the ActD peptides. The orientation of the ActD molecules at R = 2 has the quinoid portion of the phenoxazone ring at the G3pC4 site and the benzenoid portion of the phenoxazone ring at the G5pC6 site on the basis of NOE cross-peaks from ActD H7 and H8 to G5H8 and C6H6. All base pairs retain Watson-Crick type H-bonding, unlike echinomycin complexes [e.g., Gao, X., & Patel, D.J. (1988) Biochemistry 27, 1744-1751] where Hoogsteen base pairs have been observed. In contrast to previous studies on ActD, we were able to distinguish the two peptide chains.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thermodynamics of interaction of a fluorescent DNA oligomer with the anti-tumour drug netropsin.

Fluorescence spectroscopy was used to study the interaction between the minor-groove-binding drug netropsin and the self-complementary oligonucleotide d(CTGAnPTTCAG)2 containing the fluorescent base analogue 2-aminopurine (nP). The binding of netropsin to this oligonucleotide causes strong quenching of the 2-aminopurine fluorescence, observed by steady-state as well as time-resolved spectroscopy. From fluorescence titrations, binding isotherms were recorded and evaluated. The parameters showed one netropsin binding site/oligonucleotide duplex and an association constant of about 10(5) M-1 at 25 degrees C, 3-4 orders of magnitude weaker than for an exclusive adenine/thymine host sequence. From the temperature dependence of the association constant the thermodynamic parameters were obtained as delta G = -29 kJ/mol, delta H = -12 kJ/mol and delta S = +55 J.mol-1.K-1 at 25 degrees C. These parameters resemble those of the interaction of poly[(dG-dC).(dG-dC)] with netropsin, indicating a mainly entropy-driven reaction. The amino group of 2-aminopurine, like that of guanine, resides in the minor groove of DNA. Therefore the relatively weak binding of netropsin to d(CTGAnPTTCAG)2 is probably related to partial blockage of the tight fit of netropsin into the preferred minor groove of an exclusive adenine/thymine host sequence.     

Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C5-C6-T7-T8) duplex containing a pair of adjacent (G3-G4).(C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3).(C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6).(G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5).(A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G).(C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T).(A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) greater than Zn(II) greater than Cd(II) complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-dimensional 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex and a quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex

Assignment of the 1H and 31P NMR spectra of a decamer oligodeoxyribonucleotide duplex, d(CCCGATCGGG), and its quinoxaline ((MeCys3, MeCys7]TANDEM) drug duplex complex has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. The 31P chemical shifts of this 10 base pair oligonucleotide follow the general observation that the more internal the phosphate is located within the oligonucleotide sequence, the more upfield the 31P resonance occurs. While the 31P chemical shifts show sequence-specific variations, they also do not generally follow the Calladine "rules" previously demonstrated. 31P NMR also provides a convenient monitor of the phosphate ester backbone conformational changes upon binding of the drug to the duplex. Although the quinoxaline drug, [MeCys3, MeCys7]TANDEM, is generally expected to bind to duplex DNA by bis-intercalation, only small 31P chemical shift changes are observed upon binding the drug to duplex d(CCCGATCGGG). Additionally, only small perturbations in the 1H NMR and UV spectra are observed upon binding the drug to the decamer, although association of the drug stabilizes the duplex form relative to the other states. These results are consistent with a non-intercalative mode of association of the drug. Modeling and molecular mechanics energy minimization demonstrate that a novel structure in which the two quinoxaline rings of the drug binds in the minor groove of the duplex is possible.     

Inhibition of the thermally driven B to Z transition by intercalating drugs

Poly(dG-m5dC) in phosphate buffer containing 50 mM NaCl and Mg2+ will undergo a reversible thermally driven conversion from the B to the left-handed Z conformation. The temperature at the midpoint of the thermally driven B to Z transition (denoted Tz) is dependent upon the total Mg2+ concentration, with [d(1/Tz)]/(d ln [Mg]) = 0.0134 K-1. The Mg2+ concentration at the midpoint of the equilibrium B to Z transition curve, denoted [Mg]1/2, is dependent on temperature, with (d ln [Mg]1/2)/(d ln T) = -1.02. Binding of the anticancer drug daunomycin to the polymer results in a pronounced increase in Tz, dependent on the molar ratio of added drug. Tz is increased by 71.9 degrees C with nearly saturating amounts of drug bound. Transition profiles are biphasic at less than saturating amounts of bound drug. By experiments monitoring such biphasic curves at a visible wavelength sensitive to the binding of daunomycin, it may be demonstrated that no drug is released until the later phase of the transition. These results are analogous to the effects of intercalating drugs on the thermal denaturation of DNA and indicate that drug molecules preferentially interact with B-form DNA and are redistributed to regions in the B conformation over the course of the transition. Comparative studies show that some intercalators stabilize right-handed DNA more effectively than others. At similar initial binding ratios, the following order, from most to least effective, was experimentally observed: actinomycin greater than daunomycin greater than ethidium greater than proflavin.