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.     

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.     

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.     

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.     

Complete assignment of the imino protons of Escherichia coli valine transfer RNA: two-dimensional NMR studies in water

The imino proton spectrum of Escherichia coli valine tRNA has been studied by two-dimensional nuclear Overhauser effect spectroscopy (NOESY) in H2O solution. The small nuclear Overhauser effects from the imino proton of an internal base pair to the imino protons of each nearest neighbor can be observed as off-diagonal cross-peaks. In this way most of the sequential NOE connectivity trains for all the helices in this molecule can be determined in a single experiment. AU resonances can be distinguished from GC resonances by the AU imino NOE to the aromatic adenine C2-H, thus leading to specific base-pair assignments. In general, the NOESY spectrum alone is not capable of assigning every imino proton resonance even in well-resolved tRNA spectra. Multiple proton peaks exhibit more than two cross-peaks, resulting in ambiguous connectivities, and coupling between protons with similar chemical shifts produces cross-peaks that are incompletely resolved from the diagonal. The sequence of the particular tRNA determines the occurrence of the latter problem, which can often be solved by careful one-dimensional experiments. The complete imino proton assignments of E. coli valine tRNA are presented.     

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

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.     

The octamer motif in immunoglobulin genes: extraction of structural constraints from two-dimensional NMR studies.

Phase-sensitive two-dimensional nuclear Overhauser enhancement (2D NOE) and double-quantum-filtered correlated (2QF-COSY) spectra were recorded at 500 MHz for the DNA duplex d(CATTTGCATC).d(GATGCAAATG), which contains the octamer element of immunoglobulin genes. Exchangeable and nonexchangeable proton resonances including those of the H5' and H5" protons were assigned. Overall, the decamer duplex adopts a B-type DNA conformation. Scalar coupling constants for the sugar protons were determined by quantitative simulations of 2QF-COSY cross-peaks. These couplings are consistent with a two-state dynamic equilibrium between a minor N- and a major S-type conformer for all residues. The pseudorotation phase angle P of the major conformer is in the range 117-135 degrees for nonterminal pyrimidine nucleotides and 153-162 degrees for nonterminal purine nucleotides. Except for the terminal residues, the minor conformer comprises less than 25% of the population. Distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities with the MARDIGRAS algorithm confirm the dependence of the sugar pucker on pyrimidine and purine bases. Averaging by fast local motions has at most small effects on the NOE-derived interproton distances.     

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).     

Conversions of poly(2-aminodeoxyadenylate-5-halodeoxyuridylate) from B to A forms in high salt. An NMR and circular dichroism study

Proton one- and two-dimensional nuclear Overhauser enhancement (1D and 2D NOE) spectroscopy has been used to demonstrate that poly(d2NH2A-d5IU) and poly(d2NH2A-d5BrU) are converted from the B to the A conformation in high salt, as found previously for poly(d2NH2A-dT) [Borah, B., Cohen, J. S., Howard, F. B., & Miles, H. T. (1985) Biochemistry 24, 7456-7462]. The 2D NOE and 1D NOE spectra exhibit strong base proton (H8,H6)-H3' cross relaxation, suggesting short interproton distances. These results are indicative of a C3'-endo sugar pucker for both purine and pyrimidine residues in an A or closely related structure. The circular dichroism and UV spectra are consistent with the interpretation of an A conformation in high salt.     

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.     

DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix

Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond.     

500-MHz 1H NMR study of poly(dG).poly(dC) in solution using one-dimensional nuclear Overhauser effect

Secondary structures of poly(dG).poly(dC) and poly(dG).poly(dm5C) in solution are determined by nuclear Overhauser effect (NOE) measurements on GH8-deuterated and -nondeuterated DNAs with low presaturation pulse lengths (10-25 ms) and low-power and prolonged accumulations in the range of 50,000-72,000 scans. Under these conditions, the NOE difference spectra were free from diffusion. Primary NOEs between base protons GH8/CH6 and sugar protons H1', H2'/H2', and H3' suggest that in poly(dG).poly(dC) both guanine and cytosine nucleotides adopt a C3'-endo, low anti X = 200-220 degrees conformation. Computer modeling of the NOE data enable identification for the first time, in terms of the geometry of the nucleotide repeat, handedness, and helix geometry, of the structure of poly(dG).poly(dC) to be the A form, and the derived structure for the polymer duplex is very close to the single crystal structure of the double-helical d-GGGGCCCC [McCall, M., Brown, T., & Kennard, O. (1985) J. Mol. Biol. 183, 385-396]. Similar nuclear Overhauser effect data on poly(dG).poly(dm5C) revealed that G and m5C adopt a C2'endo, anti X = 240-260 degrees conformation, which indicates that this DNA exhibits the B form in solution. In summary, the results presented in this paper demonstrate that methylation of cytosines in poly(dG).poly(dC) causes A----B transition in the molecule.     

NMR studies of a deoxyribodecanucleotide containing an extrahelical thymidine surrounded by an oligo(dA).oligo(dT) tract

One- and two-dimensional NMR experiments were carried out on a decamer, d-(CGCTTTTCGC).d(GCGAAAAGCG), and on the same sequence with the addition of an unpaired thymidine, d(CGCTTTTCGC).d(GCGAATAAGCG), which will be referred to as the T-bulge decamer. Evidence from one-dimensional NOE experiments on the exchangeable protons indicates that the unpaired thymidine is extrahelical. This conclusion is also supported by numerous cross-peaks in the two-dimensional NOESY spectrum of the nonexchangeable protons. Assignments for all of the resonances, with the exception of the H5' and H5" resonances, have been made for both oligonucleotide duplexes through the use of 2D NOESY, COSY, and relayed COSY experiments. Temperature dependence of the methyl resonance chemical shifts indicates that the unpaired thymidine shows unusual behavior compared to other thymidines in the duplex. Two-dimensional NOESY experiments carried out from 5 to 35 degrees C indicate the unpaired thymidine remains extrahelical throughout this temperature range. A similar temperature dependence for the methyl chemical shift is found in the corresponding single-strand d(GCGAATAAGCG). The oligo-(dA).oligo(dT) tracts in both the decamer and the T-bulge decamer have structures different from B-form DNA and exhibit NOEs similar to those observed in other oligonucleotides containing A.T tracts. The formation of this unusual A.T tract structure may induce the extrahelical conformation of the unpaired thymidine.     

Solid-phase synthesis and high-resolution NMR studies of two synthetic double-helical RNA dodecamers: r(CGCGAAUUCGCG) and r(CGCGUAUACGCG)

Ten-micromole solid-phase RNA synthesis has been successfully performed on an automated nucleic acid synthesizer with coupling efficiencies up to 99%, using the tert-butyldimethylsilyl group to protect the 2'-hydroxyl. The tert-butyldimethylsilyl group was easily removed by tetrabutylammonium fluoride under conditions in which virtually no 2'- to 3'-isomerization was found to occur. By use of this approach, the self-complementary RNA dodecamers r(CGCGAAUUCGCG) and r(CGCGUAUACGCG) were synthesized on an automated nucleic acid synthesizer, purified by TLC, and studied by high-resolution NMR. Imino protons were assigned from one-dimensional nuclear Overhauser effects. The nonexchangeable base, H1', and H2' protons were assigned by the sequential NOESY connectivity method. The NOE data from these two oligomers were analyzed qualitatively and compared to the ideal A- and B-type helix models of Arnott et al. (1972a,b). The internucleotide H6/H8 NOEs to the preceding H1' in r(CGCGUAUACGCG) were found to be sequence-dependent and probably reflect the roll angles between adjacent bases. The internucleotide H6/H8 to H2' NOEs of these oligomers correspond very well to an A-type conformation, but the interstrand adenine H2 NOEs to the following H1' were much stronger than those predicted from the fiber model. These srong interstrand NOEs can be rationalized by base pair slide to favor more interstrand base overlap, as predicted by Callidine and Drew (1984).     

Two-dimensional 1H NMR of gene 5 protein indicates that only two aromatic rings interact significantly with oligodeoxynucleotide bases

The interaction of gene 5 protein (G5P) with oligodeoxynucleotides is investigated by 1H NMR methods, principally two-dimensional nuclear Overhauser effect spectroscopy (NOESY). Aromatic resonances of G5P are specifically assigned from crystallographic data, while the low-field resonances of nucleotides are assigned with sequential or other procedures. Chemical shift changes that accompany binding of d(pA)4, d(A)4, d(pT)4, and d(pA)8, combined with specific protein-nucleotide nuclear Overhauser effects (NOEs) obtained from NOESY spectra, suggest that Phe-73 and Tyr-26 are the only aromatic residues that stack significantly with nucleotide bases. Chemical shift data also imply a role for Leu-28, though this has not been confirmed with intermolecular NOEs. Binding of all four oligonucleotides causes marked upfield movements (0.1-0.6 ppm) of G5P NOESY cross peaks belonging to Tyr-26, Leu-28, and Phe-73. Most other G5P spin systems, notably those of Tyr-34 and Tyr-41, do not appear to be significantly affected. In the d(pA)4-G5P complex an intermolecular NOE is observed between Tyr-26 and H1' of Ade-1, while Phe-73 has NOEs with the H2, H8, and H1' protons of Ade-2 and -3. Intramolecular NOEs seem to follow a similar pattern in the partially cooperative d(pA)8-G5P complex, though specific nucleotide resonance assignments are not possible in this case. Binding causes relatively small chemical shift changes for the base resonances in adenylyl nucleotides, suggesting that there is some, but not complete, unstacking of the bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

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.     

400 MHz two-dimensional nuclear Overhauser spectroscopy on anesthetic interaction with lipid bilayer

Interaction between a volatile anesthetic, methoxyflurane, and dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was analyzed by nuclear Overhauser effect (NOE) difference spectroscopy and two-dimensional nuclear Overhauser spectroscopy (NOESY). The NOE difference spectra were obtained by selectively irradiating methoxy protons (hydrophobic end) of the anesthetic: a negative nuclear Overhauser effect of -2.94% was observed with the choline methyl protons of DPPC. The NOESY spectra revealed a cross-peak between the anesthetic methoxy protons and the choline methyl protons. A dipole-dipole interaction exists between the hydrophobic end of the anesthetic and the hydrophilic head group of DPPC. No other cross-peaks were observed. The anesthetic orients itself at the membrane/water interface by interacting with the hydrophilic surface of the DPPC membrane, leaving the hydrophilic end of the anesthetic molecule in the aqueous phase. The preferred residence site of dipolar volatile anesthetics is the membrane/water interface.