Sequence-specific interaction of Hoechst 33258 with the minor groove of an adenine-tract DNA duplex studied in solution by 1H NMR spectroscopy.

The interaction of Hoechst 33258 with the minor groove of the adenine-tract DNA duplex d(CTTTTGCAAAAG)2 has been studied in both D2O and H2O solutions by 1D and 2D 1H NMR spectroscopy. Thirty-one nuclear Overhauser effects between drug and nucleotide protons within the minor groove of the duplex, together with ring-current induced perturbations to the chemical shifts of basepair and deoxyribose protons, define the position and orientation of the bound dye molecules. Two drug molecules bind cooperatively and in symmetry related orientations at the centre of the 5'-TTTT and 5'-AAAA sequences with the binding interactions spanning only the four A-T basepairs. The positively charged N-methylpiperazine moieties point towards the centre of the duplex while the phenol groups are disposed towards the 3'-ends of the sequence. Resonance averaging is apparent for both the D2/D6 and D3/D5 phenol protons and D2"'/D6"' and D3"'/D5"' of the N-methylpiperazine ring and is consistent with these groups being involved in rapid rotation or ring-flipping motions in the bound state. Interstrand NOEs between adenine H2s and deoxyribose H1' are consistent with a high degree of propeller twisting of the A-T basepairs at the binding site of the aromatic benzimidazole and phenol rings of Hoechst. The data imply that the minor groove is particularly narrow with many contacts between the complementary curved surfaces of the drug and DNA indicating that strong van der Waals interactions, involving the floor and the walls of the minor groove, stabilize the complex. In our model the NH groups of the benzimidazole rings are positioned to make a pair of bifurcated hydrogen bonds with the adenine N3 and thymine O2 on the floor of the minor groove.     

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.     

1H NMR studies on the interaction between distamycin A and a symmetrical DNA dodecamer

High-resolution NMR techniques have been used to examine the structural and dynamical features of the interaction between distamycin A and the self-complementary DNA dodecamer duplex d-(CGCGAATTCGCG)2. The proton resonances of d(CGCGAATTCGCG)2 have been completely assigned by previous two-dimensional NMR studies [Hare, D. R., Wemmer, D. E., Chou, S. H., Drobny, G., & Reid, B. R. (1983) J. Mol. Biol. 171, 319-336]. Addition of the asymmetric drug molecule to the symmetric dodecamer leads to the formation of an asymmetric complex as evidenced by a doubling of DNA resonances over much of the spectrum. In two-dimensional exchange experiments, strong cross-peaks were observed between uncomplexed DNA and drug-bound DNA resonances, permitting direct assignment of many drug-bound DNA resonances from previously assigned free DNA resonances. Weaker exchange cross-peaks between formerly symmetry related DNA resonances indicate that the drug molecule flips head-to-tail on one duplex with half the frequency at which it leaves the DNA molecule completely. In experiments performed in H2O, nuclear Overhauser effects (NOEs) were observed from each drug amide proton to an adenine C2H and a pyrrole H3 ring proton. In two-dimensional nuclear Overhauser experiments performed on D2O solutions, strong intermolecular NOEs were observed between each of the three pyrrole H3 resonances of the drug and an adenine C2H resonance, with weaker NOEs observed between the drug H3 resonances and C1'H resonances. The combined NOE data allow us to position the distamycin A unambiguously on the DNA dodecamer, with the drug spanning the central AATT segment in the minor groove.     

Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin

We have investigated intermolecular interactions and conformational features of the netropsin complexes with d(G1-G2-A3-A4-T5-T6-C7-C8) duplex (AATT 8-mer) and the d(G1-G2-T3-A4-T5-A6-C7-C8) duplex (TATA 8-mer) by one and two-dimensional NMR studies in solution. We have assigned the amide, pyrrole and methylene 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. The directionality of the observed distance-dependent NOEs demonstrates that the 8-mer helices remain right-handed and that the arrangement of concave and convex face protons of netropsin are retained in the complexes. The observed changes in NOE patterns and chemical shift changes on complex formation suggest small conformational changes in the nucleic acid at the AATT and TATA antibiotic binding sites and possibly the flanking G.C base pairs. We observe intermolecular NOEs 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.T5 base pairs of the AATT 8-mer and TATA 8-mer duplexes. The concave face pyrrole protons of the antibiotic also exhibit NOEs to the sugar H1' protons of residues 5 and 6 in the AATT and TATA 8-mer complexes. We also detect intermolecular NOEs between the guanidino and propioamidino methylene protons at either end of netropsin and the adenosine H2 proton of the two flanking A3.T6 base pairs in the AATT 8-mer and T3.A6 base pairs in the TATA 8-mer duplexes. These studies establish a set of nine contacts between the concave face of the antibiotic and the minor groove AATT segment and TATA segment of the 8-mer duplexes in solution. The observed magnitude of the NOEs require that there be no intervening water molecules sandwiched between the concave face of the antibiotic and the minor groove of the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. The observed differences in the netropsin amide proton chemical shifts in the AATT 8-mer and TATA 8-mer complexes suggest differences in the strength and/or type of intermolecular hydrogen bonds at the AATT and TATA binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

NMR and computational characterization of mitomycin cross-linked to adjacent deoxyguanosines in the minor groove of the d(T-A-C-G-T-A).d(T-A-C-G-T-A) duplex

Two-dimensional homonuclear and heteronuclear NMR and minimized potential energy calculations have been combined to define the structure of the antitumor agent mitomycin C (MC) cross-linked to deoxyguanosines on adjacent base pairs in the d(T1-A2-C3-G4-T5-A6).d(T7-A8-C9-G10-T11-A12) duplex. The majority of the mitomycin and nucleic acid protons in the MC-X 6-mer complex have been assigned from through-bond and through-space two-dimensional proton NMR studies in aqueous solution at 5 and 20 degrees C. The C3.G10 and G4.C9 base pairs are intact at the cross-link site and stack on each other in the complex. The amino protons of G4 and G10 resonate at 9.36 and 8.87 ppm and exhibit slow exchange with solvent H2O. The NMR experimental data establish that the mitomycin is cross-linked to the DNA through the amino groups of G4 and G10 and is positioned in the minor groove. The conformation of the cross-link site is defined by a set of NOEs between the mitomycin H1" and H2" protons and the nucleic acid imino and amino protons of G4 and the H2 proton of A8 and another set of NOEs between the mitomycin geminal H10" protons and the nucleic acid imino and amino protons of G10 and the H2 proton of A2. Several phosphorus resonances of the d(T-A-C-G-T-A) duplex shift dramatically on mitomycin cross-link formation and have been assigned from proton-detected phosphorus-proton two-dimensional correlation experiments. The proton chemical shifts and NOEs establish fraying at the ends of the d(T-A-C-G-T-A) duplex, and this feature is retained on mitomycin cross-link formation. The base-base and base-sugar NOEs exhibit similar patterns for symmetry-related steps on the two nucleic acid strands in the MC-X 6-mer complex, while the proton and phosphorus chemical shifts are dramatically perturbed at the G10-T11 step on cross-link formation. The NMR distance constraints have been included in minimized potential energy computations on the MC-X 6-mer complex. These computations were undertaken with the nonplanar five-membered ring of mitomycin in each of two pucker orientations. The resulting low-energy structures MX1 and MX2 have the mitomycin cross-linked in a widened minor groove with the chromophore ring system in the vicinity of the G10-T11 step on one of the two strands in the duplex.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sequence specific molecular recognition and binding by a GC recognizing Hoechst 33258 analogue to the decadeoxyribonucleotide d-[CATGGCCATG]2: structural and dynamic aspects deduced from high field 1H-NMR studies.

The non-exchangeable and imino proton NMR resonances have been assigned of the 1:1 complex of an analogue 2 of Hoechst 33258 1 bound to the decadeoxyribonuycleotide d-[CATGGCCATG]2 by a combination of NOE difference, COSY and NOESYPH techniques. In contrast to Hoechst 33258 which recognizes 5'-AATT sequences exclusively, analogue 2 possesses structural features designed to permit the recognition of GC sites. The NOESY and 1D-NOE experiments place the drug in the minor groove and it is located on the 5'-CCAT sequence. The orientation of the drug in the groove is such as to place the N-methylpiperazine terminus at a GC site. Cross-correlation peaks in the NOESY experiment show that the DNA duplex retains its right-handed B form, similar to that in the free decamer. Specific NOEs locate the benzoxazole moiety on the 5'-CCAT and are consistent with the pyridine nitrogen forming a new hydrogen bond to G(4)-2NH2 at 5'-CCAT. The drug appears to undergo rotation around the C9-C10 bond, at a rate comparable with NMR time scale, even after binding. Variable temperature 1H-NMR studies established that the DNA is thermally stabilized as a result of the drug binding. The drug binding is a dynamic process involving exchange between the equivalent 5'-CCAT sites at approximately 60s-1 with delta G degree of 65 kJ mol-1 at 308K. The experimental evidence is in accord with a slide-swing mechanism for this process.     

Molecular recognition and binding of a GC site-avoiding thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: 1H-NMR evidence for thiazole intercalation

The structural and dynamic aspects of the interaction of the thiazole containing lexitropsin (1) with an oligodeoxyribonucleotide were studied by high field 1H-NMR spectroscopy. Complete assignment of the 1H-NMR resonances of lexitropsin 1 was accomplished by 2D-NMR techniques. The complexation-induced chemical shifts and NOE cross peaks in the NOESY map of the 1:1 complex of lexitropsin (1) and d-[CGCAATTGCG]2 reveal that the thiazole ring of the lexitropsin (1) intercalates between dA4.A5 bases and the rest of the ligand resides in the minor groove of the AT rich core of decamer, thus occupying the 5'-AATT sequence on the DNA. Intercalation of the thiazole moiety of the drug has been detected by the presence of intermolecular NOEs both in the major and the minor groove of the decamer helix. The absence of intranucleotide NOEs between base protons and H1'/H2' protons suggested local unwinding of the binding site on the DNA. From COSY and NOESY methods of 2D-NMR, it was established that the N-formyl (amino) terminus of the thiazole lexitropsin (1) is projecting into the major groove towards A5H8 while the amidinium terminus lies in the minor groove towards the T7G8 base pairs of the opposite strand. The expected intranucleotide NOEs confirmed that the decadeoxyribonucleotide in the 1:1 complex exists in a right handed B-conformation. The presence of exchange signals along the binding site 5'-AATT indicated an exchange of the bound drug process wherein the rate of exchange between the two equivalent sites was estimated to be congruent to 130 s-1 at 30 degrees C and with delta G degrees of 62.4 kJ mol-1. Force field and Pi calculations permitted a rationalization of the experimentally observed binding mode in terms of preferred conformation of the ligand and repeat length in lexitropsins compared with the DNA receptor.     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.     

Structural and dynamic aspects of binding of a prototype lexitropsin to the decadeoxyribonucleotide d(CGCAATTGCG)2 deduced from high-resolution 1H NMR Studies

Structural and dynamic properties of the self-complementary decadeoxyribonucleotide d(CGCAATTGCG)2 and the interaction between a prototype lexitropsin, or information-reading oligopeptide, and the decadeoxyribonucleotide are deduced by using high-resolution 1H NMR techniques. The nonexchangeable and imino proton resonances of d(CGCAATTGCG)2 have been completely assigned by two-dimensional NMR studies. The decadeoxyribonucleotide exists as a right-handed B-DNA. In the 1H NMR spectrum of the 1:1 complex, the selective chemical shifts and removal of degeneracy of AH2(4), AH2(5), T-CH3(6), and T-CH3(7) due to the anisotropy effects of the heterocyclic moieties of the ligand, and with lesser effects at the flanking base sites C(3) and G(8), locate the drug centrally in the decadeoxyribonucleotide. This conclusion is supported by plots of individual chemical shift changes across the decadeoxyribonucleotide. Similarly, imino protons IV and V experience larger shifts and II and III smaller shifts in accord with this conclusion while drug complexation permits the detection of imino proton I. Strong nuclear Overhauser effects (NOEs) between pyrrole H5 and AH2(5), and weaker NOEs to AH1'(5), TH3'(6), and AH2'(5), firmly locate the ligand in the minor groove. Intraligand NOEs between the adjacent heterocyclic moieties indicate that the lexitropsin is subject to propeller twisting about the N6-C9 bond in both the bound and free forms. Nuclear Overhauser effect spectroscopy (NOESY) and correlated spectroscopy (COSY) experiments also indicate that the removal of degeneracy of the C16 methylene protons upon complexation may arise from restricted rotation about the C15-N9, C15-C16, and C16-C17 bonds. Specific hydrogen bonds between amide NH groups on the concave face of the ligand (N4H, N6H, N9H) and adenine N3 or thymine O2 on the floor of the minor groove are in accord with displacement of the hydration shell by the drug. NOE measurements on the decadeoxyribonucleotide in the 1:1 complex confirm it exists as a right-handed helix and belongs to the B family. Exchange NMR effects permit an estimate of a rate of approximately equal to 44 s-1 for the two-site exchange of the lexitropsin between two equivalent sites on the decamer with delta G++ approximately equal to 70 +/- 5 kJ mol-1 at 294 K. Alternative mechanisms for this exchange process are considered.     

Structural analysis of d(GCAATTGC)2 and its complex with berenil by nuclear magnetic resonance spectroscopy

The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.     

Structural and dynamic aspects of non-intercalative (1:1) binding of a thiazole-lexitropsin to the decadeoxyribonucleotide d-[CGCAATTGCG]2: An 1H-NMR and molecular modeling study.

The location, orientation and dynamics of a thiazole-containing analogue of distamycin 1 bound to the decadeoxyribonucleotide d-[CGCAATTGCG]2 have been studied by non-exchangable and imino proton NMR resonances of the 1:1 complex. Using NOE difference, COSY and NOESY experiments, lexitropsin (1) was located in the minor groove of DNA at 5'-CAAT sequence. This was concluded by an intermolecular NOE between the ligand and a minor groove A4H2 proton. The NOE cross-correlations in the NOESY map confirmed that the DNA decamer duplex in the 1:1 complex remains in a right-handed B-conformation similar to that in the free decamer. Experiments on non-exchangeable and exchangeable proton NMR resonances placed the N-formylamino terminus of drug 1 on the 5'-C3 nucleotide, while the rest of the molecule extends onto the 5'-AAT sequence. The structural evidence for sequence preferential binding at 5'CAAT rather than 5'AATT suggests this reflects an attempt on the part of the sterically demanding inward directed sulfur of the thiazole to minimize compression by moving part of the molecule to the somewhat wider CG base site. The lack of evidence for a 2:1 drug:DNA complex, in contrast to distamycin, is in accord with this interpretation. The lexitropsin 1 was found to be in an exchange between the equivalent 5'-CAAT sites at a rate of approximately 35S-1 with a delta G degree of 65 +/- 5 kJ mol-1 at 303 K. The experimental data suggests a slide-swing mechanism for this exchange process.     

Two-dimensional NMR studies of d(GGTTAATGCGGT).d(ACCGCATTAACC) complexed with the minor groove binding drug SN-6999.

The dodecadeoxynucleotide duplex d(GGTTAATGCGGT).d(ACCGCATTAACC) and its 1:1 complex with the minor groove binding drug SN-6999 have been prepared and studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments have been obtained for the free duplex by standard methods. The line widths of the resonances in the complex are greater than those observed for the free duplex, which complicates the assignment process. Extensive use of two-quantum spectroscopy was required to determine the scalar correlations for identifying all of the base proton and most of the 1'H-2'H-2'H spin subsystems for the complex. This permitted unambiguous sequence-specific resonance assignments for the complex, which provides the necessary background for a detailed comparison of the structure of the duplex, with and without bound drug. A series of intermolecular NOEs between drug and DNA were identified, providing sufficient structural constraints to position the drug in the minor groove of the duplex. However, the combination of NOEs observed can only be rationalized by a model wherein the drug binds in the minor groove of the DNA in both orientations relative to the long helix axis and exchanges rapidly between the two orientations. The drug binds primarily in the segment of five consecutive dA-dT base pairs d(T3T4A5A6T7).d(A18T19T20A21A22), but surprisingly strong interactions are found to extend one residue in the 3' direction along each strand to G8 and C23. The observation of intermolecular contacts to residues neighboring the AT-rich region demonstrates that the stabilization of the bis(quaternary ammonium) heterocycle family of AT-specific, minor groove binding drugs is not based exclusively on interactions with dA-dT base pairs.     

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence-dependent recognition of DNA duplexes: netropsin complexation to the TATA site of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

We have recorded one-dimensional exchangeable proton and two-dimensional nonexchangeable proton nmr spectra on the complex of netropsin with the self-complementary d(G-G-T-A-T-A-C-C) duplex in aqueous solution between 25° and 35°C. The antibiotic amide, pyrrole, and methylene protons, and the nucleic acid base and sugar H1′, H2′, H2″, and H3′ protons, have been assigned from an analysis of the two-dimensional nuclear Overhauser effect (NOESY) spectra of the complex. We observe intermolecular NOEs between the antibiotic concave face amide, pyrrole, and CH₂ resonances, and the adenosine H2 and sugar H1′ protons of base-pairs T3·A6 and A4·T5 in the central TATA core of the d(G₁-G₂-T₃-A₄-T₅-A₆-C₇-C₈) duplex. We present a molecular model outlining these seven antibiotic-DNA contacts for the complex in solution. The observed line-broadening of several base and sugar protons at the TATA minor groove netropsin binding site in the complex at 35°C are interpreted in terms of intermediate exchange between two orientations of bound netropsin on the duplex.     

Structure and dynamics of ligand-template interactions of topoisomerase inhibitory analogs of Hoechst 33258: high field 1H-NMR and restrained molecular mechanics studies.

The binding characteristics of Hoechst 33258 (1), a synthetic bis-benzimidazole, and its structural analog 2, with one of the benzimidazoles replaced by a pyridoimidazole, to the self-complementary decadeoxyribonucleotide sequences d(CGCAATTGCG)2 (A) and d-(CATGGCCATG)2 (B) respectively, were examined using high field 1H-NMR techniques. Selective complexation induced chemical shift changes, the presence of exchange signals and intermolecular NOE contacts between the ligands and the minor groove protons of the oligonucleotides suggest the preferred binding sites as the centrally located AATT segment for complex A1, and the CCAT segment for complex B2. The B-type conformations of the two DNA duplexes are preserved upon complexation, as confirmed by the 2D-NOESY based sequential connectivities involving DNA base and sugar protons. Close intermolecular NOE based contacts between the ligands and their respective DNA sequences were further refined to model the ligand-DNA complexes starting from the computer generated B-type structures for the oligonucleotides. Force field calculations of ligand-DNA interaction energies indicate a more favorable contribution from the van der Waals energy component in the case of complex A1 consistent with its stronger net binding compared with the complex B2. Overall, the incorporation of a pyridinic nitrogen in Hoechst 33258 structure alters its selectivity for base pair recognition from A.T to G.C, resulting largely from the formation of a hydrogen bond between the new basic center and the 2-NH2 group of a guanosine moiety. The rates for the exchange of ligands between the two equivalent binding sites (AATT for 1, and CCAT for 2) of the self-complementary DNA sequences, are estimated from analyses of coalescence of NMR signals to be 189s-1 at 301 K for A1 and 79s-1 at 297 K for B2; which correspond to delta G++ of 13.8 and 18.6 kcal.mol-1 respectively.     

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].     

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.     

Evidence for DAPI intercalation in CG sites of DNA oligomer [d(CGACGTCG)]2: a 1H NMR study.

The interaction between 4',6-diamidino-2-phenylindole (DAPI) and the DNA oligomer [d(CGACGTCG)]2 has been investigated by proton one- and two-dimensional NMR spectroscopy in solution. Compared with the minor groove binding of the drug to [d(GCGATCGC)]2, previously studied by NMR spectroscopy, the interaction of DAPI with [d(CGACGTCG)]2 appears markedly different and gives results typical of a binding mechanism by intercalation. C:G imino proton signals of the [d(CGACGTCG)]2 oligomer as well as DAPI resonances appear strongly upfield shifted and sequential dipolar connectivities between cytosine and guanine residues show a clear decrease upon binding. Moreover, protons lying in both the minor and major grooves of the DNA double helix appear involved in the interaction, as evidenced principally by intermolecular drug-DNA NOEs. In particular, the results indicate the existence of two stereochemically non-equivalent intercalation binding sites located in the central and terminal adjacent C:G base pairs of the palindromic DNA sequence. Different lifetimes of the complexes were also observed for the two sites of binding. Moreover, due to the fast exchange on the NMR timescale between free and bound species, different interactions in dynamic equilibrium with the observed intercalative bindings were not excluded.     

Solution structure of the chromomycin-DNA complex

The structure of the chromomycin-DNA complex at the deoxyoctanucleotide duplex level has been determined from one- and two-dimensional proton NMR studies in Mg-containing aqueous solution. The NMR results demonstrate that the antitumor agent binds as a symmetrical dimer to the self-complementary d[T-T-G-G-C-C-A-A] duplex with retention of the 2-fold symmetry in the complex. A set of intermolecular nuclear Overhauser enhancements (NOEs) establishes that two chromomycin molecules in the dimer share the minor groove at the G-G-C-C.G-G-C-C segment in such a way that each hydrophilic edge of the chromophore is located next to the G-G.C-C half-site and each C-D-E trisaccharide chain extends toward the 3'-direction of the octanucleotide duplex. In addition, the A-B disaccharide segment and the hydrophilic side chain of the antitumor agent are directed toward the phosphate backbone. The observed changes in nucleic acid NOEs and coupling patterns on complex formation establish a transition to a wider and shallower minor groove at the central G-G-C-C.G-G-C-C segment required for accommodating the chromomycin dimer. The present demonstration that chromomycin binds as a dimer and switches the conformation of the DNA at its G.C-rich minor groove binding site provides new insights into antitumor agent design and the sequence specificity of antitumor agent-DNA recognition.