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.     

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.     

Three-dimensional homonuclear NOESY-TOCSY of an intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple: nonexchangeable proton assignments and structural implications.

Two- and three-dimensional homonuclear 1H NMR spectroscopic techniques have been applied to obtain nearly complete nonexchangeable proton assignments for a 31-residue intramolecular pyrimidine.purine.pyrimidine DNA triplex containing a central G.TA triple in D2O. An assignment strategy for obtaining resonance assignments for DNA protons from a 3D NOESY-TOCSY spectrum is proposed. The strategy utilizes the H1'/H5 omega 3 planes and relies on the recognition of cross-peak patterns for obtaining both intraresidue as well as sequential assignments. On the basis of the cross-peaks observed in the 2D and 3D spectra, a few structural features of the triplex have been delineated qualitatively. All three strands of the triplex adopt a right-handed helical conformation, and, despite the introduction of a central purine guanosine, there is no evidence for major structural distortions in the protonated third strand on the basis of a qualitative interpretation of NMR data. Several interstrand contacts between the purine and the Hoogsteen pyrimidine strands are observed which define the relative orientation of the bases and sugars in these two strands. The presence of strong NOEs between the methyl protons of thymine and the H1' proton of guanosine defines the preferred base-pairing alignment of guanosine at the G.TA triple site. The general approaches illustrated in this study extend the range of DNA molecules accessible for detailed structural investigation by high-resolution NMR spectroscopy.     

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.     

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.     

Structure of DNA hexamer sequence d-CGATCG by two-dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics

Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5' d-CpG 3' site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5'/H5" protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N<-->S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by approximately 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is approximately 180 degrees and approximately 120 degrees, respectively while all other residues are close to C2'endo-conformation. A large propeller twist (approximately -18 degrees) and smallest twist angle (approximately 31 degrees) at A3pT4 step, in the middle of the sequence, a wider (12 A) and shallower (3.0 A) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding.     

The differences in the T2 relaxation rates of the protons in the partially-deuteriated and fully protonated sugar residues in a large oligo-DNA ('NMR-window') gives complementary structural information.

Selective incorporation of the stereospecifically deuteriated sugar moieties (> 97 atom % 2H enhancements at H2', H2', H3' and H5'/5' sites, approximately 85 atom % 2H enhancement at H4' and approximately 20 atom % 2H enhancement at H1') in DNA and RNA by the 'NMR-window' approach has been shown to solve the problem of the resonance overlap [refs. 1, 2 & 3]. Such specific deuterium labelling gives much improved resolution and sensitivity of the residual sugar proton (i.e. H1' or H4') vicinal to the deuteriated centers (ref. 3). The T2 relaxation time of the residual protons also increases considerably in the partially-deuteriated (shown by underline) sugar residues in dinucleotides [d(CpG), d(GpC), d(ApT), d(TpA)], trinucleotide r(A2'p5'A2'p5'A) and 20-mer DNA duplex 5'd(C1G2C3-G4C5G6C7G8A9A10T11T12C13G14C15G16C17G18C19G20)(2) 3'. The protons with shorter T2 can be filtered away using a number of different NMR experiments such as ROESY, MINSY or HAL. The NOE intensity of the cross-peaks in these experiments includes only straight pathway from H1' to aromatic proton (i-i and i-i + 1) without any spin-diffusion. The volumes of these NOE cross-peaks could be measured with high accuracy as their intensity is 3 to 4 times larger than the corresponding peaks in the fully protonated residues in the normal NOESY spectra. The structural informations thus obtainable from the residual protons in the partially-deuteriated part of the duplex and the fully protonated part in the 'NMR window' can indeed complement each other.     

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.     

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.     

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)     

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.     

1H NMR and optical spectroscopic investigation of the sequence-dependent dimerization of a symmetrical cyanine dye in the DNA minor groove

A symmetrical cyanine dye was previously shown to bind as a cofacial dimer to alternating A-T sequences of duplex DNA. Indirect evidence suggested that dimerization of the dye occurred in the minor groove. 1H NMR experiments reported here verify this model based on broadening and shifting of signals due to protons on carbon 2 of adenine and imino protons at the central five A-T pairs of the 11 base pair duplex: 5'-GCGTATATGCG-3'/3'-CGCATATACGC-5'. This binding mode is similar to that of distamycin A, even though the dye lacks the hydrogen-bonding groups used by distamycin for sequence-specific recognition. Surprisingly, the third base pair (G-C) was also implicated in the binding site. UV-vis experiments were used to compare the extent of dimerization of the dye for 11 different sequence variants. These experiments verified the importance of a G-C pair at the third position: replacing this pair with A-T suppressed dimerization. These results indicate that the dye binding site spans six base pairs: 5'-GTATAT-3'. The initial G-C pair seems to be important for widening the minor groove rather than for making important contacts with the dye molecules since inverting its orientation to C-G or replacing it with I-C still led to favorable dimerization of the dye.     

Solution structure of a trans-opened (10S)-dA adduct of (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in a fully complementary DNA duplex: evidence for a major syn conformation

Two-dimensional NMR was used to determine the solution structure of an undecanucleotide duplex, d(CGGTCACGAGG).d(CCTCGTGACCG), in which (+)-(7S,8R,9S,10R)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene is covalently bonded to the exocyclic N(6)() amino group of the central deoxyadenosine, dA(6), through trans addition at C10 of the epoxide (to give a 10S adduct). The present study represents the first NMR structure of a benzo[a]pyrene (10S)-dA adduct in DNA with a complementary T opposite the modified dA. Exchangeable and nonexchangeable protons of the modified duplex were assigned by the use of TOCSY (in D(2)O) and NOESY spectra (in H(2)O and D(2)O). Sequential NOEs expected for a B-type DNA conformation with typical Watson-Crick base pairing are observed along the duplex, except at the lesion site. We observed a strong intraresidue NOE cross-peak between H1' and H8 of the modified dA(6). The sugar H2' and H2' ' of dC(5) lacked NOE cross-peaks with H8 of dA(6) but showed weak interactions with H2 of dA(6) instead. In addition, the chemical shift of the H8 proton (7.51 ppm) of dA(6) appears at a higher field than that of H2 (8.48 ppm). These NOE and chemical shift data for the dA(6) base protons are typical of a syn glycosidic bond at the modified base. Restrained molecular dynamics/energy minimization calculations show that the hydrocarbon is intercalated from the major groove on the 3'-side of the modified base between base pairs A(6)-T(17) and C(7)-G(16) and confirm the syn glycosidic angle (58 degrees ) of the modified dA(6). In the syn structure, a weak A-T hydrogen bond is possible between the N3-H proton of T(17) and N7 of dA(6) (at a distance of 3.11 A), whereas N1, the usual hydrogen bonding partner for N3-H of T when dA is in the anti conformation, is 6.31 A away from this proton. The 10(S)-dA modified DNA duplex remains in a right-handed helix, which bends in the direction of the aliphatic ring of BaP at about 42 degrees from the helical axis. ROESY experiments provided evidence for interconversion between the major, syn conformer and a minor, possibly anti, conformer.     

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)     

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.     

1H and 15N NMR investigation of the interaction of pyrimidine nucleotides with ribonuclease A

Extensive 1H and 15H NMR investigations of the nucleotide moieties capable of hydrogen bonding to ribonuclease A were carried out in order to gain more detailed information on the specificity of nucleotide-enzyme interaction. The 1H investigations focussed on those protons presumed to be involved in hydrogen bonding between the various nucleotides and the enzyme. In particular these were the imino protons of the uridine nucleotides and the amino protons of the cytidine nucleotides. The technique of 15N-1H double quantum filtering was applied for observation of the resonances of the latter in the nucleotide-enzyme complex. The downfield shift observed for the imino proton resonance of the uridine nucleotides was indicative of hydrogen bond formation to the enzyme. 15N NMR spectra of the free nucleotides and the nucleotide-enzyme complexes were also acquired to examine the possibility of hydrogen bond formation at the N3 site of both pyrimidine bases and the amino group of the cytidine nucleotides. The downfield shift observed for the 15N3 resonance of the uridine nucleotides and the upfield shift observed for the corresponding resonance of the cytidine nucleotides was evidence that the N3 moiety acts as hydrogen donor or hydrogen acceptor in the nucleotide-enzyme complex. The effect of complex formation on the 15N1 resonance of the respective bases was also studied. Both 1H and 15N NMR results indicated subtle differences between the complexes of the 2' and 3' nucleotides. The extent of hydrogen bonding as well as the arrangement of the nucleotide base at the active site of the enzyme varies in dependence on the position of the phosphate group. It is established that hydrogen bonding, though not the main binding force between the nucleotides and the enzyme, is certainly a major factor of RNase A specificity for pyrimidine nucleotides.     

15N labeling of oligodeoxynucleotides for NMR studies of DNA-ligand interactions.

The amino protons of 15N-labeled DNA were studied as a possible structural probe in NMR investigations of the interaction of DNA with various ligands. Since the imino protons are located in the center of the double helix, and variations of their chemical shift values are difficult to interpret in terms of structural changes, these probes are not very useful. Instead, amino protons are located in the major or minor groove of the DNA and are often directly involved in the binding of a ligand. For a selective probing 4-15NH2-2'-deoxycytidine and 6-15NH2-2'-deoxyadenosine were obtained by chemical synthesis. The labeled nucleosides were introduced in distinct positions of oligodeoxynucleotides by large-scale DNA synthesis. Direct 15N NMR and 1H-15N multiple quantum NMR were applied to detect the corresponding 15N labels or protons attached to the 15N labels. Chemical shift values for the cytidine and the adenosine amino nitrogen and proton resonances of a symmetric 18 base pair lac operator sequence are reported.     

Transcriptional enhancer related DNA sequences: anomalous 1H NMR NOE crosspeaks.

A dynamic heterogeneity which correlates with the function of the operator DNA in the lactose operon of E. coli. was previously observed (1) as a local minimum in the thymine imino proton T1 centered at a GTG/C-CAC sequence. Since this triplet occurs frequently in DNA regulatory regions, it was proposed that these sequences may be part of a structural element for specific protein interaction. We examine here three additional biologically significant 17 base pair duplexes containing GTG/CAC triplets: (1) a sequence from the mouse heavy chain immunoglobulin enhancer, (2) a sequence from the critical core of the Simian Virus 40 (SV40) enhancer, and (3) a sequence from pBR322 plasmid used as control for experiments with the SV40 DNA sequences. The 1H NMR resonance assignment for nearly all the nonexchangeable protons for both eukaryotic enhancer duplexes with the exception of the H5'/H5" protons was accomplished to use for structural analysis of these duplexes. The data presented show several NOE's associated with the GTG/CAC triplets which suggest structural variation from uniform B-DNA. In addition, anomalous broad crosspeaks for the fixed thymine methyl to its own H6 proton in combination with the imino proton kinetics associated with these triplets reinforces the original observation of a sequence dependent dynamic variation.     

High resolution NMR study of CAP binding site 22mer in H2O solution

High resolution proton NMR were measured for the deoxyoligonucleotide 22mer duplex corresponding to the CAP (catabolite gene activator protein) binding site of lac promotor. The spectra in the lower field region than the water resonance were taken with the time-shared Redfield pulse method by using a JEOL 500 MHz NMR spectrometer. In the imino proton region 18 peaks were separately observed, but the area intensity at 10 degrees C corresponds to 20 protons. By selective irradiation at each peak position NOEs (nuclear Overhauser effects) were observed between the imino and adenine C2H protons and between imino proton themselves. By tracing sequential NOE train carefully, 17 imino proton signals could be unambiguously assigned to each base pair except five AT base pairs at terminals. With the elevation of temperature the peaks showed gradual broadening and disappeared, which indicates the stepwise base pair opening of the duplex. Referring to the above peak assignments it can be concluded that GC20 and AT4 pairs close to terminals relax first and the base pair opening proceeds toward central GC13 and 14.