Two-dimensional NMR studies on the anthramycin-d(ATGCAT)2 adduct

Two-dimensional NMR experiments were performed on the adduct of anthramycin with d(ATGCAT)2 to obtain the assignments of the nucleotide base and sugar protons as well as the anthramycin protons. Anthramycin is covalently attached to a guanine 2-amino group, forming the d(ATamGCAT).d(ATGCAT) modified duplex. The anthramycin protons in the minor groove exhibit NOEs to several nucleotide protons. The network of anthramycin-nucleotide NOEs and the measurement of the 10-Hz coupling constant between the anthramycin H11 and H11a protons shows that anthramycin is covalently attached as the S stereoisomer at the anthramycin C11 position with the side chain of anthramycin oriented toward the 5' end of the modified strand. The NOE data show that the anthramycin-modified duplex is in a right-handed conformation with all bases in an anti conformation. Analysis of the J1'-2' coupling constants for the resolved H1' resonances shows that the S-type conformation of the sugars is highly preferred.     

Minor groove hydration of DNA in aqueous solution: sequence-dependent next neighbor effect of the hydration lifetimes in d(TTAA)2 segments measured by NMR spectroscopy.

The hydration in the minor groove of double stranded DNA fragments containing the sequences 5'-dTTAAT, 5'-dTTAAC, 5'-dTTAAA and 5'-dTTAAG was investigated by studying the decanucleotide duplex d(GCATTAATGC)2 and the singly cross-linked decameric duplexes 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3' and 5'-d(GCCTTAAAGC)-3'-linker-5'-d(GCTTTAAGGC)-3' by NMR spectroscopy. The linker employed consisted of six ethyleneglycol units. The hydration water was detected by NOEs between water and DNA protons in NOESY and ROESY spectra. NOE-NOESY and ROE-NOESY experiments were used to filter out intense exchange cross-peaks and to observe water-DNA NOEs with sugar 1' protons. Positive NOESY cross-peaks corresponding to residence times longer than approximately 0.5 ns were observed for 2H resonances of the central adenine residues in the duplex containing the sequences 5'-dTTAAT and 5'-dTTAAC, but not in the duplex containing the sequences 5'-dTTAAA and 5'-dTTAAG. In all nucleotide sequences studied here, the hydration water in the minor groove is significantly more mobile at both ends of the AT-rich inner segments, as indicated by very weak or negative water-A 2H NOESY cross-peaks. No positive NOESY cross-peaks were detected with the G 1'H and C 1'H resonances, indicating that the minor groove hydration water near GC base pairs is kinetically less restrained than for AT-rich DNA segments. Kinetically stabilized minor groove hydration water was manifested by positive NOESY cross-peaks with both A 2H and 1'H signals of the 5'-dTTAA segment in d(GCATTAATGC)2. More rigid hydration water was detected near T4 in d(GCATTAATGC)2 as compared with 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3', although the sequences differ only in a single base pair. This illustrates the high sensitivity of water-DNA NOEs towards small conformational differences.     

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.     

Two-dimensional NMR investigation of a bent DNA fragment: assignment of the proton resonances and preliminary structure analysis.

The resonances of all the non-exchangeable protons (except 5'H and 5"H) of d(CGAAAAATCGG) + d(CCGATTTTTCG), a putatively bent DNA duplex, have been assigned using 1H two-dimensional nuclear magnetic resonance methods. The nuclear Overhauser effect data indicate an overall B-form structure for this double-helical DNA undecamer. However, several features of the NMR data such as some unusually weak C8/C6 proton to C1' proton NOE cross-peaks, the presence of relatively intense C2H to C1'H NOE cross-peaks, and unusual chemical shifts of some 2", 2', and 1' protons suggest a substantial perturbation of the helix structure at the junctions and along the length of the tract of A residues. These structural deviations are considered in terms of models of DNA bending.     

Sequence-dependent conformations of DNA duplexes: the TATA segment of the d(G-G-T-A-T-A-C-C) duplex in aqueous solution

The base and sugar protons of the d(G-G-T-A-T-A-C-C) duplex have been assigned from two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements in D₂O solution at 25°C. The nucleic acid protons have been assigned from NOEs between protons on adjacent bases on the same and partner strands, as well as from NOEs between the base protons and their own and 5′-flanking H1′, H2′, H2″, H3′, and H4′ sugar protons. These assignments are confirmed from coupling constant and NOE connectivities within the sugar protons of a given residue. Several of these NOEs exhibit directionality and demonstrate that the d(G-G-T-A-T-A-C-C) duplex is a right-handed helix. The relative magnitude of the NOEs between the base protons and the sugar H2′ protons of its own and 5′-flanking sugar demonstrate that the TATA segment of the d(G-G-T-A-T-A-C-C) duplex adopts a B-DNA type helix geometry in solution, in contrast to the previous observation of a A-type helix for the same octanucleotide duplex in the crystalline state.     

Alkyl phosphotriester modified oligodeoxyribonucleotides. VI. NMR and UV spectroscopic studies of ethyl phosphotriester (Et) modified Rp-Rp and Sp-Sp duplexes, (d[GGAA(Et)TTCC])2.

1H NMR chemical shift assignments for the title compounds were made for all but a few H5' and H5" signals using two-dimensional nuclear Overhauser effect (2D-NOE) data, which was also used for the first time to assign absolute configuration at phosphorus. The chemical shifts were, in general, similar to those reported [Broido, M.S., et al. (1985) Eur. J. Biochem. 150, 117-128] for the B-like conformation of the unmodified, parent duplex, [d(GGAATTCC)]2. Differences in chemical shifts for corresponding protons were mostly localized to the AA(Et)TT region, and showed some stereochemical dependence. Unambiguous assignment of the phosphotriester 31P signals was achieved in a novel way using selective insensitive nucleus enhancement by polarization transfer (selective INEPT) NMR. The Rp-Rp duplex melted ca. 11 degrees C lower than either the Sp-Sp or parent duplexes, as evidenced by Tm and variable temperature 1H/31P NMR measurements. The 2D-NOE data for the Rp-Rp duplex suggested possible steric interactions between the ethyl group and the H3' of the flanking A residue. At low ionic strength, the Sp-Sp and parent duplexes had similar stability but at high ionic strength the Sp-Sp duplex was less stable.     

Covalent carcinogenic lesions in DNA: NMR studies of O6-methylguanosine containing oligonucleotide duplexes

We report on proton and phosphorus high resolution NMR investigations of the self-complementary dodecanucleotide d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6 meG.N 12-mers), N = C, T, A and G, which contain N3.O6meG10 interactions in the interior of the helix. These sequences containing a single modified O6meG per strand were prepared by phosphoamidite synthesis and provide an excellent model for probing the structural basis for covalent carcinogenic lesions in DNA. Distance dependent nuclear Overhauser effect (NOE) measurements and line widths of imino protons demonstrate that the N3 and O6meG.10 bases stack into the duplex and are flanked by stable Watson-Crick base pairs at low temperature for all four O6meG.N 12-mer duplexes. The imino proton of T3 in the O6meG.T 12-mer and G3 in the O6meG.N 12-mer helix, which are associated with the modification site, resonate at unusually high field (8.5 to 9.0 ppm) compared to imino protons in Watson-Crick base pairs (12.5 to 14.5 ppm). The nonexchangeable base and sugar protons have been assigned from two dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) measurements on the O6meG.N 12-mer helices. The directionality of the distance dependent NOEs establish all O6meG.N duplexes to be right-handed helices in solution. The glycosidic torsion angles are in the anti range at the N3.O6meG10 modification site except for O6meG10 in the O6meG.G 12-mer duplex which adopts a syn configuration. This results in altered NOEs between the G3 (anti).O6meG10 (syn) pair and flanking G2.C11 and G4.C9 base pairs in the O6meG.G 12-mer duplex. We observe pattern reversal for cross peaks in the COSY spectrum linking the sugar H1' protons with the H2',2" protons at the G2 and O6meG10 residues in the O6meG.N 12-mer duplexes with the effect least pronounced for the O6meG.T 12-mer helix. The proton chemical shift and NOE data have been analyzed to identify regions of conformational perturbations associated with N3.O6meG10 modification sites in the O6meG.N 12-mer duplexes. The proton decoupled phosphorus spectrum of O6meG.T 12-mer duplex exhibits an unperturbed phosphodiester backbone in contrast to the phosphorus spectra of the O6meG.C 12-mer, O6meG.G 12-mer and O6meG.A 12-mer duplexes which exhibit phosphorus resonances dispersed over 2 ppm characteristic of altered phosphodiester backbones at the modification site. Tentative proposals are put forward for N3.O6meG10 pairing models based on the available NMR data and serve as a guide for the design of future experiments.     

High-resolution NMR study of a synthetic DNA-RNA hybrid dodecamer containing the consensus pribnow promoter sequence: d(CGTTATAATGCG).r(CGCAUUAUAACG)

The nonsymmetrical double-helical hybrid dodecamer d(CGTTATAATGCG).r(CGCAUUAUAACG) was synthesized with solid-phase phosphoramidite methods and studied by high-resolution 2D NMR. The imino protons were assigned by one-dimensional nuclear Overhauser methods. All the base protons and H1', H2', H2", H3', and H4' sugar protons of the DNA strand and the base protons, H1', H2', and most of the H3'-H4' protons of the RNA strand were assigned by 2D NMR techniques. The well-resolved spectra allowed a qualitative analysis of relative proton-proton distances in both strands of the dodecamer. The chemical shifts of the hybrid duplex were compared to those of the pure DNA double helix with the same sequence (Wemmer et al., 1984). The intrastrand and cross-strand NOEs from adenine H2 to H1' resonances of neighboring base pairs exhibited characteristic patterns that were very useful for checking the spectral assignments, and their highly nonsymmetric nature reveals that the conformations of the two strands are quite different. Detailed analysis of the NOESY and COSY spectra, as well as the chemical shift data, indicate that the RNA strand assumes a normal A-type conformation (C3'-endo) whereas the DNA strand is in the general S domain but not exactly in the normal C2'-endo conformation. The overall structure of this RNA-DNA duplex is different from that reported for hybrid duplexes in solution by other groups (Reid et al., 1983a; Gupta et al., 1985) and is closer to the C3'-endo-C2'-endo hybrid found in poly(dA).poly(dT) and poly(rU).poly(dA) in the fiber state (Arnott et al., 1983, 1986).     

Sequential NMR assignments of labile protons in DNA using two-dimensional nuclear-Overhauser-enhancement spectroscopy with three jump-and-return pulse sequences

Two-dimensional nuclear Overhauser enhancement (NOESY) spectra of labile protons were recorded in H2O solutions of a protein and of a DNA duplex, using a modification of the standard NOESY experiment with all three 90 degree pulses replaced by jump-and-return sequences. For the protein as well as the DNA fragment the strategically important spectral regions could be recorded with good sensitivity and free of artifacts. Using this procedure, sequence-specific assignments were obtained for the imino protons, C2H of adenine, and C4NH2 of cytosine in a 23-base-pair DNA duplex which includes the 17-base-pair OR3 repressor binding site of bacteriophage lambda. Based on comparison with previously published results on the isolated OR3 binding site, these data were used for a study of chain termination effects on the chemical shifts of imino proton resonances of DNA duplexes.     

Conformational changes in the oligonucleotide duplex d(GCGTTGCG) x d(CGCAACGC) induced by formation of a cis-syn thymine dimer. A two-dimensional NMR study

In order to obtain insight into the repair mechanism of DNA containing thymine photo-dimer, the conformation of the duplex d(GCGTTGCG) x d(CGCAACGC) with a thymine dimer incorporated has been studied by proton NMR and the results are compared with NMR data of the parent octamer. Two-dimensional nuclear Overhauser enhancement (2D NOE) spectroscopy and two-dimensional homonuclear Hartmann-Hahn spectroscopy have been applied to assign all the non-exchangeable base protons and most of the deoxyribose protons of both duplexes. From these experiments it is clear that indeed a cis-syn cyclobutane-type thymine photodimer is formed by the irradiation of this oligonucleotide with ultraviolet light. Comparison of 2D NOE spectra and the 1H chemical shifts of the damaged and the intact DNA duplexes reveals that formation of a thymine dimer induces small distortions of the B-DNA structure, the main conformational change occurring at the site of the thymine dimer.     

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.     

Hydration of DNA in aqueous solution: NMR evidence for a kinetic destabilization of the minor groove hydration of d-(TTAA)2 versus d-(AATT)2 segments.

The residence times of the hydration water molecules near the base protons of d-(GTGGAATTCCAC)2 and d-(GTGGTTAACCAC)2 were investigated by nuclear magnetic resonance (NMR) spectroscopy. Nuclear Overhauser effects (NOE) were observed between base protons of the DNA and hydration water in NOESY and ROESY experiments. Large positive NOESY cross peaks observed between the resonances of the water and the adenine 2H protons of the central d-(AATT)2 segment in the duplex d-(GTGGAATTCCAC)2 indicate the presence of a 'spine of hydration' with water molecules exhibiting residence times on the DNA longer than 1 nanosecond. In contrast, no positive intermolecular NOESY cross peaks were detected in the d-(TTAA)2 segment of the duplex d-(GTGGTTAACCAC)2, indicating that no water molecules bound with similarly long residence times occur in the minor groove of this fragment. These results can be correlated with the larger width of the minor groove in d-(TTAA)2 segments as compared to that in d-(AATT)2 segments, as observed previously in single crystal structures of related oligonucleotide duplexes in B type conformation. The present experiments confirm earlier experimental results from single crystal studies and theoretical predictions that a 5'-dTA-3' step in the nucleotide sequence interrupts the spine of hydration in the minor groove.     

Determination of the major tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA adduct by 1H and 15N NMR studies

(+)-CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of the drug are thought to be due to the formation of a covalent adduct with DNA through N3 of adenine. Although the covalent linkage sites between (+)-CC-1065 and DNA have been determined, the tautomeric form of the covalently modified adenine in the (+)-CC-1065-DNA duplex adduct was not defined. A [6-15N]deoxyadenosine-labeled 12 base pair non-self-complementary oligomer, d(GGCGGAGTT*AGG).d(CCTAACTCCGCC) (asterisk indicates 15N-labeled base), containing the (+)-CC-1065 most preferred binding sequence 5'AGTTA, was synthesized and modified with (+)-CC-1065. This [6-15N]deoxyadenosine-labeled 12-mer duplex adduct was then studied by 1H and 15N NMR. One-dimensional NOE difference and two-dimensional NOESY 1H NMR experiments on the nonisotopically labeled 12-mer duplex adduct demonstrate that the 6-amino protons of the covalently modified adenine exhibit two signals at 9.19 and 9.08 ppm. Proton NMR experiments on the [6-15N]deoxyadenosine-labeled 12-mer duplex adduct show that the two resonance signals for adenine H6 observed on the nonisotopically labeled duplex adduct were split into doublets by the 15N nucleus with coupling constants of 91.3 Hz for non-hydrogen-bonded and 86.8 Hz for hydrogen-bonded amino protons. Parallel 15N NMR experiments on the [6-15N]deoxyadenosine-labeled (+)-CC-1065-12-mer duplex adduct show a triplet-like signal around -276.9 ppm and coupling constants of 91.5 and 85.6 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

NMR structure of a parallel-stranded DNA duplex at atomic resolution

DNA dodecamers have been designed with two cytosines on each end and intervening A and T stretches, such that the oligomers have fully complementary A:T base pairs when aligned in the parallel orientation. Spectroscopic (UV, CD and IR), NMR and molecular dynamics studies have shown that oligomers having the sequences d(CCATAATTTACC) and d(CCTATTAAATCC) form a parallel-stranded duplex when dissolved at 1:1 stoichiometry in aqueous solution. This is due to the C:C⁺ clamps on either end and extensive mismatches in the antiparallel orientation. The structure is stable at neutral and acidic pH. At higher temperatures, the duplex melts into single strands in a highly cooperative fashion. All adenine, cytosine and thymine nucleotides adopt the anti conformation with respect to the glycosidic bond. The A:T base pairs form reverse Watson–Crick base pairs. The duplex shows base stacking and NOEs between the base protons T(H6)/A(H8) and the sugar protons (H1′/H2′/H2″) of the preceding nucleotide, as has been observed in antiparallel duplexes. However, no NOEs are observed between base protons H2/H6/H8 of sequential nucleotides, though such NOEs are observed between T(CH₃) and A(H8). A three-dimensional structure of the parallel-stranded duplex at atomic resolution has been obtained using molecular dynamics simulations under NMR constraints. The simulated structures have torsional angles very similar to those found in B-DNA duplexes, but the base stacking and helicoid parameters are significantly different.     

Two-dimensional proton nuclear magnetic resonance investigation of the synthetic deoxyribonucleic acid decamer d(ATATCGATAT)2

In this study two-dimensional NMR techniques (COSY and NOESY) have been used in conjunction with one-dimensional NMR results to complete the assignment of the proton NMR spectrum of the double-stranded DNA decamer, d(ATATCGATAT)2, and to obtain qualitative information about numerous interproton distances in this molecule and some limited information about conformational dynamics. COSY and NOESY measurements have been combined to systematically assign many of the resonances from the H1' and H2',2" sugar protons to specific nucleotides in the double helix. This method relies on the fact that sugar protons within a specific nucleotide are scalar coupled and that base protons (AH8, GH8, TH6, and CH6) in right-handed helices can interact simultaneously with their own H2',2" sugar protons and those of the adjacent (5'-3') nucleotide attached to its 5' side (i.e., XpA not ApX). A COSY experiment is used to identify sugar resonances within a residue whereas the NOESY experiment allows the neighboring sugar to be connected (linked). The CH5 and CH6 resonances in the spectrum can immediately be identified by the COSY experiment. The methyl protons of thymine residues exhibit strong through-space interbase interactions both with their own TH6 proton and with AH8 proton on the adjacent (5'-3') adenine residue. These interactions are used both to make assignments of the spectra and to establish that the thymine methyl groups are in close proximity to the AH8 protons of adjacent adenine residues [Feigon, J., Wright, J. M., Leupin, W., Denny, W. A., & Kearns, D. R. (1982) J. Am. Chem. Soc. 104, 5540].(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent carcinogenic O6-methylguanosine lesions in DNA. Structural studies of the O6 meG X A and O6meG X G interactions in dodecanucleotide duplexes

High-resolution proton and phosphorus nuclear magnetic resonance studies are reported on the self-complementary d(C1-G2-N3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplexes (henceforth called O6meG X A 12-mer when N3 = A3 and O6meG X G 12-mer when N3 = G3), which contain symmetry-related A3 X O6meG10 and G3 X O6meG10 interactions in the interior of the helices. We observe inter-base-pair nuclear Overhauser effects (NOE) between the base protons at the N3 X O6meG10 modification site and protons of flanking G2 X C11 and G4 X C9 base-pairs, indicative of the stacking of N3 and O6meG10 bases in both O6meG X A 12-mer and O6meG X G 12-mer duplexes. We have assigned all the base and a majority of the sugar protons from two-dimensional proton-correlated and nuclear Overhauser effect experiments on the O6meG X A 12-mer duplex and O6meG X G 12-mer duplex in solution. The observed NOEs establish that the A3 and O6meG10 at the modification site and all other residues adopt the anti configuration about the glycosidic bond, and that the O6meG X A 12-mer forms a right-handed duplex. The interaction between the bulky purine A3 and O6meG10 residues in the anti orientation results in large proton chemical shift perturbations at the (G2-A3-G4) X (C9-O6meG10-C11) segments of the helix. By contrast, we demonstrate that the O6meG10 residue adopts a syn configuration, while all other bases adopt an anti configuration about the glycosidic bond in the right-handed O6meG X G 12-mer duplex. This results in altered NOE patterns between the base protons of O6meG10 and the base and sugar protons of flanking C9 and C11 residues in the O6meG X G 12-mer duplex. The phosphorus backbone is perturbed at the modification site in both duplexes, since the phosphorus resonances are dispersed over 2 parts per million in the O6meG X A 12-mer and over 1 part per million in the O6meG X G 12-mer compared to a 0.5 part per million dispersion for an unperturbed DNA helix. We propose tentative pairing schemes for the A3 X O6meG10 and G3 X O6meG10 interactions in the above dodecanucleotide duplexes.     

Nmr studies of an extrahelical cytosine in an A·T rich region of a deoxyribodecanucleotide

One-dimensional and two-dimensional (2D) nmr experiments were carried out on an oligonucleotide duplex that contains an unpaired cytosine, d(GCGAAC AAGCG)·d(CGCTTTTCGC), which will be referred to as the C-bulge decamer. Evidence from one-dimensional nuclear Overhauser effect (NOE) experiments on the exchangeable protons indicates that the unpaired cytosine is extrahelical. This conclusion is also supported by numerous cross-peaks in the 2D NOE spectroscopy (NOESY) spectrum of the nonexchangeable protons. The assignments for all of the resonances, with the exception of the H5′ and H5″ resonances, have been made through the use of 2D NOESY, correlated spectroscopy (COSY), and relayed COSY experiments. The temperature dependence of the C(H6) resonance chemical shifts indicates that the unpaired cytosine shows unusual behavior compared to other cytosines in the duplex. A comparison of chemical shifts for all, the assigned resonances of the duplexes with and without the unpaired cytosine suggests that the majority of the structural perturbation is localized in the A·T tract surrounding the unpaired base. The behavior of the imino resonances as a function of temperature also indicates that the perturbation to the duplex is localized and destabilizes the A·T base pairs adjacent to the unpaired base. © 1993 John Wiley & Sons, Inc.     

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.     

Pyrimidine.pyrimidine base-pair mismatches in DNA. A nuclear magnetic resonance study of T.T pairing at neutral pH and C.C pairing at acidic pH in dodecanucleotide duplexes

Structural features of pyrimidine.pyrimidine mismatches in the interior of oligonucleotide duplexes have been investigated by high resolution two-dimensional proton nuclear magnetic resonance (n.m.r.) spectroscopy. These studies were conducted on the self-complementary d(C-G-C-T-A-G-C-T-T-G-C-G) duplex (designated T.T 12-mer) and the self-complementary d(C-G-C-C-A-G-C-T-C-G-C-G) duplex (designated C.C 12-mer) containing T.T and C.C pairs located at identical positions four base-pairs from either end of the duplex. Proton n.m.r. studies on the T.T 12-mer duplex were undertaken in the neutral pH range, while studies on the C.C 12-mer duplex were recorded at acidic pH. The proton spectra narrowed considerably on lowering the pH below neutrality for the C.C 12-mer duplex. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) data sets have been recorded on the T.T 12-mer and C.C 12-mer duplexes in high salt H2O and D2O solution. The magnitude of the NOE crosspeaks and the directionality of the NOE connectivities demonstrate that both duplexes are right-handed with all bases, including those at the mismatch site, adopting an anti configuration about the glycosidic bond. The observed base and sugar proton chemical shifts suggest structural similarities for the trinucleotide segments centered about the T.T and C.C mismatches. A NOE is detected between the resolved imino protons of T4 and T9 at the mismatch site, consistent with formation of a stacked "wobble" T4(anti).T9(anti) pair in the T.T 12-mer duplex. A comparison of the imino proton chemical shift and NOE data suggests that the imino-carbonyl hydrogen bonds in the wobble T.T mismatch are weaker than the corresponding imino-carbonyl hydrogen bonds in the wobble G.T mismatch. The 4-amino protons of C4 and C9 at the mismatch site in the C.C 12-mer duplex do not exhibit the pattern of hydrogen-bonded and exposed protons separated by approximately 1.5 parts per million characteristic of cytidine amino protons involved in Watson-Crick G.C pairing. The experimental data are insufficient to differentiate between wobble C(anti).C+(anti) and other pairing possibilities for the mismatch in the C.C 12-mer duplex at acidic pH.