NMR conformational analysis of p-tolyl furanopyrimidine 2'-deoxyribonucleoside and crystal structure of its 3',5'-di-O-acetyl derivative

The conformation of a representative molecule of a new, potent class of antiviral-active modified nucleosides is determined. A bicyclic nucleoside, 3-(2'-deoxy-beta-D-ribofuranosyl)-6-(4-methylphenyl)-2,3-dihydrofuro[2,3-d]pyrimidin-2-one, shows C2'-endo and C3'-endo ribose conformations in solution (63:37, 37 degrees C; DMSO-d6), as determined by 1H NMR studies. The crystal structure of a 3',5'-di-O-acetyl-protected derivative (monoclinic, P21, a/b/c= 6.666(1)/12.225(1)/24.676(2) A, beta=90.24(1) degrees , Z=4) shows exclusively C2'-endo deoxyribose puckering. The base is found in the anti position both in solution and in crystalline form.     

Conformational microheterogeneity in a DNA double helix: structure of restriction endonuclease Bam H1 recognition site

Structural studies using 500 MHz 1H NMR spectroscopy on Bam H1 recognition site d(GGATCC)2 in solution at 19 degrees is reported. The resonances from the sugar ring and base protons have been assigned from the 2D-COSY and NOESY spectra. Analyses of the NOESY cross-peaks between the base protons H8/H6 and sugar protons H2'/H2", H3' reveal that the nucleotide units G2, A3 and C6 adopt (C3'-endo, chi = 200 degrees-220 degrees) conformation while G1, T4 and C5 exhibit (C2'-endo, chi = 240 degrees-260 degrees) conformation. NMR data clearly suggest that the two strands of d(GGATCC)2 are conformationally equivalent and there is a structural two-fold between the two A-T pairs. The above information and the NOESY data are used to generate a structural model of d(GGATCC)2. The important features are: (i) G1-G2 stack, the site of cleavage, shows an alternation in sugar pucker i.e. C2'-endo, C3'-endo as in a B-A junction, (ii) G2-A3 stack adopts a mini A-DNA, both the sugars being C3'-endo, (iii) A3-T4 stack, the site of two-fold, displays an A-B junction with alternation in sugar pucker as C3'-endo, C2'-endo, (iv) T4-C5 stack adopts a mini B-DNA both the sugars being C2'-endo and (v) C5-C6 stack exhibits a B-A junction with C2'-endo, C3'-endo sugar puckers. Thus, our studies demonstrate that conformational microheterogeneity with a structural two fold, is present in the Bam H1 recognition site.     

Determination of the DNA sugar pucker using 13C NMR spectroscopy

Solid-state 13C NMR spectroscopy of a series of crystalline nucleosides and nucleotides allows direct measurement of the effect of the deoxyribose ring conformation on the carbon chemical shift. It is found that 3'-endo conformers have 3' and 5' chemical shifts significantly (5-10 ppm) upfield of comparable 3'-exo and 2'-endo conformers. The latter two conformers may be distinguished by smaller but still significant differences in the carbon chemical shifts at the C-2' and C-4' positions. High-resolution solid-state NMR of three modifications of fibrous calf thymus DNA shows that these trends are maintained in high-molecular-weight DNA and confirms that the major ring pucker in A-DNA is 3'-endo, while both B-DNA and C-DNA are largely 2'-endo. The data show that 13C NMR spectroscopy is a straightforward and useful probe of DNA ring pucker in both solution and the solid state.     

Conformational studies of d-(AAAAATTTTT)2 using constraints from nuclear overhauser effects and from quantitative analysis of the cross-peak fine structures in two-dimensional 1H nuclear magnetic resonance spectra

The conformation at the dA-dT junction in d-(AAAAATTTTT)2 was investigated by using a variety of phase-sensitive two-dimensional nuclear magnetic resonance experiments at 500 MHz for detailed studies of the deoxyribose ring puckers. Conformational constraints were collected from two-dimensional nuclear Overhauser enhancement spectra recorded with short mixing times and from quantitative simulations of the cross-peaks in two-dimensional correlated spectra. Overall, the decamer duplex adopts a conformation of the B-DNA type, and for dA4 and dA5 the pseudorotation phase angle P is in the standard range 150-180 degrees. The deoxyribose puckers for the other nucleotides deviate significantly from the standard B-DNA structure. Spectrum simulations assuming either static deviations from standard B-DNA or a simple two-state dynamic equilibrium between the C2'-endo and C3'-endo forms of the deoxyribose were used to analyze the experimental data. It was thus found that the ring pucker for dT6 deviates from the regular C2'-endo form of B-DNA by a static distortion, with the pseudorotation phase angle P in the range 100-130 degrees, and a similar value of P is indicated for dT7. For the peripheral base pairs dynamic distortions of the C2'-endo form of the deoxyribose were found. In agreement with recent papers on related duplexes containing (dA)n tracts, we observed prominent nuclear Overhauser effects between adenine-2H and deoxyribose-1'H, which could be largely due to pronounced propeller twisting as observed in the crystal structures of (dA)n-containing compounds.     

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Structural analysis of 2',3'-dideoxyinosine, 2',3'-dideoxyadenosine, 2',3'-dideoxyguanosine and 2',3'-dideoxycytidine by 500-MHz 1H-NMR spectroscopy and ab-initio molecular orbital calculations.

Solution structure of anti-AIDS drug, 2',3'-dideoxyinosine (ddI) has been assessed by NMR spectroscopy and pseudorotational analysis in conjunction with its analogues: 2',3'-dideoxyadenosine (ddA), 2',3'-dideoxyguanosine (ddG) and 2',3'-dideoxycytidine (ddC). The absence of 3'-hydroxyl groups in these compounds has prompted us to establish the relationship between proton-proton and corresponding endocyclic torsion angles in the 2',3'-dideoxyribofuranose moiety on the basis of five available crystal structures of 2',3'-dideoxynucleosides. A subsequent pseudorotational analysis on ddI (1), ddA (2), ddG (3) and ddC (4) shows that the twist C2'exo-C3'-endo forms of sugar are overwhelmingly preferred (75-80%) over the C2'-endo envelope forms. The phase angles (P) for North and South conformers with the corresponding puckering amplitude (psi m) for ddI (1), ddA (2) and ddG (3) are as follows: PN = 0.1 degrees, PS = 161 degrees and psi m = 34.1 degrees for ddI (1); PN = 1.4 degrees, PS = 160 degrees and psi m = 34.2 degrees for ddA (2) and PN = 2.4 degrees, PS = 163 degrees and psi m = 33.6 degrees for ddG (3). The predominant North conformer of ddC (4) is intermediate between twist C2'-exo-C3'-endo and C3'-endo envelope (P = 10.9 degrees) with a psi m of 34.7 degrees. Note that these preponderant North-sugar structures (approx. 75-80%) found in the solution studies of ddI (1), ddA (2), dG (3) and ddC (4) are not reflected in the X-ray crystal structures of 2',3'-dideoxyadenosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures denosine and 2',3'-dideoxycytidine. The constituent sugar residues in both of these crystal structures are found to be in the South-type geometry (ddA crystalizes in C3'-exo envelope form, while ddC adopts the form intermediate between the C3'-exo envelope and C3'-endo-C4'-exo twist form). This means that X-ray structures of ddA (2) and ddC (4) only represent the minor conformer of the overall pseudorotamer population in solution. An assumption that the structure of the pentofuranose sugar (i.e. P and psi m) participating in conformational equilibrium described by the two-state model remains unchanged at different temperatures has been experimentally validated by assessing five unknown pseudorotational parameters with eight unique observables (3J1'2', 3J1'2", 3J2'3', 3J2'3", 3J2"3', 3J2"3", 3J3'4' and 3J3"4') for 2',3'-dideoxynucleosides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deoxyribose ring conformation of [d(GGTATACC)]2: an analysis of vicinal proton-proton coupling constants from two-dimensional proton nuclear magnetic resonance

Exchangeable and nonexchangeable protons of [d(GGTATACC)]2 in aqueous cacodylate solution were assigned from two-dimensional nuclear Overhausser effect (2D NOE) spectra. With phase-sensitive COSY and double quantum filtered COSY (DQF-COSY) experiments, the cross-peaks resulting from deoxyribose ring conformation sensitive proton-proton vicinal couplings, i.e., all 1'-2', 1'-2", 2'-3', and 3'-4' couplings and six from 2"-3' couplings, were observed. From the cross-peak fine structure, the 2',2" proton assignments can be confirmed; coupling constants J1'2' and J1'2" and sums of coupling constants involving H2' and H2" for all residues and H3' for C8 were obtained. The DISCO procedure [Kessler, H., Muller, A., & Oschkinat, H. (1985) Magn. Reson. Chem. 23, 844-852] was used to extract individual 1'-2' and 1'-2" coupling constants. The sum of coupling constants involving H1' or H3' was measured from the one-dimensional spectrum where signal overlap is not a problem. Analysis of the resulting coupling constants and sums of coupling constants, in the manner of Rinkel and Altona [Rinkel, L. J., & Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649], led to the following conclusion: C2'-endo deoxyribose ring conformation is predominant for every residue, but a significant amount of C3'-endo conformation may exist, ranging from 14% to 30%.     

Molecular structures of two new anti-HIV nucleoside analogs: 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine and 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine.

The x-ray crystal structures of two new anti-HIV compounds, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)adenine (2'-F-dd-araA) and 9-(2,3-dideoxy-2-fluoro-beta-D-threo- pentofuranosyl)hypoxanthine (2'-F-dd-aral), have been determined at two temperatures. Both crystals are in the space group P2(1)2(1)2(1), and their structures were solved by direct methods. Least-squares refinement produced final R-factors of 0.027 for the 2'-F-dd-araA structure and of 0.044 for the 2'-F-dd-aral structure, respectively. The latter structure contains a two-fold disordered conformation of the sugar moiety. All three conformers (one for 2'-F-dd-araA and two for 2'-F-dd-aral) adopt an anti chi CN glycosyl torsion angle. The sugar in the 2'-F-dd-araA structure has a C2'-endo pucker conformation, whereas the sugar in the 2'-F-dd-aral structure has a mixture of C2'-endo and C3'-endo pucker conformations. When the sugar adopts the C2'-endo conformation, the torsion angle about the C4'-C5' bond is in a transgauche+ conformation. In contrast, when the sugar adopts the C3'-endo conformation, the torsion angle about the C4'-C5' bond is in a gauche(+)-gauche- conformation. The C2'-F bond distance is 1.406(3) A, similar to that found in other aliphatic C-F bonds. The results suggest that the 2'-fluoro-2',3'-dideoxyarabinosyl nucleosides do not have a strong preference for either C2'-endo or C3'-endo sugar pucker.     

Conformational analysis of d(C3G3), a B-family duplex in solution

NMR and circular dichroism studies of the duplex formed by the self-complementary DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs from standard B-form. An analysis of NMR coupling constants within the deoxyribose moieties yields a 70% or greater contribution from pseudorotation phase angles corresponding to the C3'-exo conformation, a conformation similar to the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton distances are consistent with a B-form structure, but some internucleotide distances are intermediate between A- and B-form structures. Circular dichroism spectra have B-form characteristics but also include an unusual negative band at 282 nm. The solution spectroscopic results are in contrast with X-ray crystallographic studies which find A-form structures for similar sequences.     

1H NMR study of the sugar pucker of 2',3'-dideoxynucleosides with anti-human immunodeficiency virus (HIV) activity

The sugar ring conformations of 2',3'-dideoxyribosyladenine (ddA), 2',3'-dideoxyribosylcytosine (ddC), 2',3'-dideoxyribosylguanine (ddG), 2',3'-dideoxyribosylhypoxanthine (ddI), 3'-azido-2',3'-dideoxyribosylthymine (AZT), 3'-azido-2',3'-dideoxyribosyluracil (AZU) and 3'-fluoro-2',3'-dideoxyribosylthymine (FddT) have been investigated by 1H NMR spectroscopy. While the sugar ring in FddT exists almost totally in C2'-endo geometry, other nucleosides show equilibrium between sugar puckers of C3'-endo family (N-type) and C2'-endo family (S-type). For unsubstituted dideoxynucleosides C3'-endo conformer is favoured (congruent to 75%), whereas for AZT and AZU both the conformers have almost equal populations. Unlike X-ray diffraction studies, the NMR results do not support the suggestion that C3'-exo sugar puckers are desirable for the anti-HIV activity of these nucleosides.     

NMR studies of fully modified locked nucleic acid (LNA) hybrids: solution structure of an LNA:RNA hybrid and characterization of an LNA:DNA hybrid

LNA is a bicyclic nucleic acid analogue that contains one or more 2'-O,4'-C methylene linkage(s), which effectively locks the furanose ring in a C3'-endo conformation. We report here the NMR solution structure of a nonamer LNA:RNA hybrid and a structural characterization of a nonamer LNA:DNA hybrid, where the LNA strands are composed entirely of LNA nucleotides. This is the first structural characterization of fully modified LNA oligonucleotides. The high-resolution structure reveals that the LNA:RNA hybrid adopts an almost canonical A-type duplex morphology. The helix axis is almost straight and the duplex geometry is regular. This shows that fully modified LNA oligomers can hybridize with complementary RNA and form duplexes within the Watson-Crick framework. The LNA:DNA hybrid structurally resembles an RNA:DNA hybrid as shown by determination of deoxyribose sugar puckers and analysis of NOESY NMR spectra.     

Molecular structure, conformation and interactions of antitumor antibiotic cyanonaphthridinomycin, a covalent binder of DNA

X-ray, NMR and molecular modeling studies on cyanonaphthridinomycin (C22H26N4O5), a DNA binding antibiotic, have been carried out to study the structure, conformation and interactions with DNA. The crystals belong to the space group P21 with the cell dimensions of a = 5.934(1)b = 20.684(4), c = 16.866(3)A, gamma = 90.9 degrees and Z = 4(two molecules/asymmetric unit). The structure was solved by direct methods and difference Fourier methods and refined to an R value of 0.087 for 4061 reflections. The conformation of the molecule is compared with that of naphthridinomycin. There are differences in the orientation of the methoxyl group and the saturated oxazole ring. 1 and 2D NMR studies have been carried out and the dihedral angles obtained from coupling constants have been compared with those obtained from the crystal structure. Molecular mechanics studies were carried out to obtain the energy minimized structure and its comparison with X-ray and NMR results. Molecular modelling studies were performed to propose models for drug-DNA interactions. Both partial intercalation and groove-binding models have been proposed.     

Conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d (5''-GCATGC)2 complex studied in solution by 1H-n.m.r. spectroscopy.

The conformation and dynamics of the deoxyribose rings of a (nogalamycin)2-d(5'-GCATGC)2 complex have been determined from an analysis of 1H-1H vicinal coupling constants and sums of coupling constants (J1'-2',J1'-2",epsilon 1', epsilon 2' and epsilon 2") measured from one-dimensional n.m.r. spectra and from H-1'-H-2' and H-1'-H-2" cross-peaks in high-resolution phase-sensitive two-dimensional correlation spectroscopy (COSY) and double-quantum-filtered correlation spectroscopy (DQF-COSY) experiments. The value of J3'-4' has also been estimated from the magnitude of H-3'-H-4' cross-peaks in DQF-COSY spectra and H-1'-H-4' coherence transfer cross-peaks in two-dimensional homonuclear Hartman-Hahn spectroscopy (HOHAHA) spectra. The data were analysed, in terms of a dynamic equilibrium between North (C-3'-endo) and South (C-2'-endo) conformers, by using the graphical-analysis methods described by Rinkel & Altona [(1987) J. Biomol. Struct. Dyn. 4,621-649]. The data reveal that the sugars of the 2C-5G and 3A-4T base-pairs, which form the drug-intercalation site, have strikingly different properties. The deoxyribose rings of the 2C-5G base-pair are best described in terms of an equilibrium heavily weighted in favour of the C-2'-endo geometry (greater than 95% 'S'), with a phase angle, P, lying in the range 170-175 degrees and amplitude of pucker between 35 and 40 degrees, as typically found for B-DNA. For the deoxyribose rings of the 3A-4T base-pair, however, the analysis shows that, for 3A, the C-2'-endo and C3'-endo conformers are equally populated, whereas a more limited data set for the 4T nucleotide restricts the equilibrium to within 65-75% C-2'-endo. The deoxyribose rings of the 1G-6C base-pair have populations of 70-80% C-2'-endo, typical of nucleotides at the ends of a duplex. Although drug-base-pair stacking interactions are an important determinant of the enhanced duplex stability of the complex [Searle, Hall, Denny, & Wakelin (1988) Biochemistry 27, 4340-4349], the current findings make it clear that the same interactions can be associated with considerable variations in the degree of local structural dynamics at the level of the sugar puckers.     

NMR study of stacking interactions and conformational adjustments in the dinucleotide-carcinogen adduct 2'-deoxycytidylyl-(3----5)-2'-deoxy-8-(N-fluoren-2-ylac etamido)guanosi ne

The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.     

Molecular structure of cyclic deoxydiadenylic acid at atomic resolution

The molecular structure of a small cyclic nucleotide, cyclic deoxydiadenylic acid, has been determined by single-crystal X-ray diffraction analysis and refined to an R factor of 7.8% at 1.0-A resolution. The crystals are in the monoclinic space group C2 with unit cell dimensions of a = 24.511 (3) A, b = 24.785 (3) A, c = 13.743 (3) A, and beta = 94.02 (2) degrees. The structure was solved by the direct methods program SHELXS-86. There are 2 independent cyclic d(ApAp) molecules, 2 hydrated magnesium ions, and 26 water molecules in the asymmetric unit of the unit cell. The two cyclic d(ApAp) molecules have similar conformations within their 12-membered sugar-phosphate backbone ring, but they have quite different appearances due to the different glycosyl torsion angles that make one molecule more compact and the other extended and open. Three of the four deoxyribose rings are in the less common C3'-endo conformation. All four phosphate groups have their phosphodiester torsion angles alpha/zeta in the gauche(+)/gauche(+) conformation. One of the cyclic d(ApAp) molecules associates with another symmetry-related molecule to form a self-intercalated dimer that is a stable structure in solution, as observed in NMR studies. Many interesting intermolecular interactions, including base-base stacking, ribose-base stacking, base pairing, base-phosphate hydrogen bonding, and metal ion-phosphate interactions, are found in the crystal lattice. This structure may be relevant for understanding the conformational potentiality of an endogenous biological regulator of cellulose synthesis, cyclic (GpGp).     

An oligodeoxyribonucleotide N3'--> P5' phosphoramidate duplex forms an A-type helix in solution.

The solution conformations of the dinucleotide d(TT) and the modified duplex d(CGCGAATTCGCG)2 with N3'--> P5' phosphoramidate internucleoside linkages have been studied using circular dichroism (CD) and NMR spectroscopy. The CD spectra indicate that the duplex conformation is similar to that of isosequential phosphodiester RNA, a A-type helix, and is different from that of DNA, a B-type helix, NMR studies of model dimers d(TpT) and N3'--> P5' phosphoramidate d(TnpT) show that the sugar ring conformation changes from predominantly C2'-endo to C3'-endo when the 3'-phosphoester is replaced by a phosphoramidate group. Two-dimensional NMR (NOESY, DQF-COSY and TOCSY spectra) studies of the duplex provide additional details about the A-type duplex conformation of the oligonucleotide phosphoramidate and confirm that all furanose rings of 3'-aminonucleotides adopt predominantly N-type sugar puckering.     

Crystal structure of the topoisomerase II poison 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide bound to the DNA hexanucleotide d(CGTACG)2.

The structure of the complex formed between d(CGTACG)(2) and the antitumor agent 9-amino-[N-(2-dimethylamino)ethyl]acridine-4-carboxamide has been solved to a resolution of 1.6 A using X-ray crystallography. The complex crystallized in space group P6(4) with unit cell dimensions a = b = 30.2 A and c = 39.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The asymmetric unit contains a single strand of DNA, 1. 5 drug molecules, and 29 water molecules. The final structure has an overall R factor of 19.3%. A drug molecule intercalates between each of the CpG dinucleotide steps with its side chain lying in the major groove, and the protonated dimethylamino group partially occupies positions close to ( approximately 3.0 A) the N7 and O6 atoms of guanine G2. A water molecule forms bridging hydrogen bonds between the 4-carboxamide NH and the phosphate group of the same guanine. Sugar rings adopt the C2'-endo conformation except for cytosine C1 which moves to C3'-endo, thereby preventing steric collision between its C2' methylene group and the intercalated acridine ring. The intercalation cavity is opened by rotations of the main chain torsion angles alpha and gamma at guanines G2 and G6. Intercalation perturbs helix winding throughout the hexanucleotide compared to B-DNA, steps 1 and 2 being unwound by 8 degrees and 12 degrees, respectively, whereas the central TpA step is overwound by 17 degrees. An additional drug molecule, lying with the 2-fold axis in the plane of the acridine ring, is located at the end of each DNA helix, linking it to the next duplex to form a continuously stacked structure. The protonated N,N-dimethylamino group of this "end-stacked" drug hydrogen bonds to the N7 atom of guanine G6. In both drug molecules, the 4-carboxamide group is internally hydrogen bonded to the protonated N-10 atom of the acridine ring. The structure of the intercalated complex enables a rationalization of the known structure-activity relationships for inhibition of topoisomerase II activity, cytotoxicity, and DNA-binding kinetics for 9-aminoacridine-4-carboxamides.     

Crystal structure of an adenine bulge in the RNA chain of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag)

Crystal structure of a DNA.RNA hybrid, d(CTCCTCTTC).r(gaagagagag), with an adenine bulge in the polypurine RNA strand was determined at 2.3 A resolution. The structure was solved by the molecular replacement method and refined to a final R-factor of 19.9% (Rfree 22.2%). The hybrid duplex crystallized in the space group I222 with unit cell dimensions, a = 46.66 A, b = 47.61 A and c = 54.05 A, and adopts the A-form conformation. All RNA and DNA sugars are in the C3'-endo conformation, the glycosyl angles in anti conformation and the majority of the C4'-C5' torsion angles in g+ except two trans angles, in conformity with the C3'-endo rigid nucleotide hypothesis. The adenine bulge is looped out and it is also in the anti C3'-endo conformation. The bulge is involved in a base-triple (C.g)*a interaction with the end base-pair (C9.g10) in the minor groove of a symmetry-related molecule. The 2' hydroxyl group of g15 is hydrogen bonded to O2P and O5' of g17, skipping the bulged adenine a16 and stabilizing the sugar-phosphate backbone of the hybrid. The hydrogen bonding and the backbone conformation at the bulged adenine site is very similar to that found in the crystal structure of a protein-RNA complex.     

Nuclear Overhauser effect studies of the conformation of Co(NH3)4ATP bound to kidney Na,K-ATPase

Transferred nuclear Overhauser effect measurements (in the two-dimensional mode) have been used to determine the three-dimensional conformation of an ATP analogue, Co(NH3)4ATP, at the active site of sheep kidney Na,K-ATPase. Previous studies have shown that Co(NH3)4ATP is a competitive inhibitor with respect to MnATP for the Na,K-ATPase [Klevickis, C., & Grisham, C.M. (1982) Biochemistry 21, 6979. Gantzer, M.L., et al. (1982) Biochemistry 21, 4083]. Nine unique proton-proton distances on ATPase-bound Co(NH3)4ATP were determined from the initial build-up rates of the cross-peaks of the 2D-TRNOE data sets. These distances, taken together with previous 31P and 1H relaxation measurements with paramagnetic probes, are consistent with a single nucleotide conformation at the active site. The bound Co(NH3)4ATP) adopts an anti conformation, with a glycosidic torsion angle of 35 degrees, and the conformation of the ribose ring is slightly N-type (C2'-exo, C3'-endo). The delta and gamma torsional angles in this conformation are 100 degrees and 178 degrees, respectively. The nucleotide adopts a bent configuration, in which the triphosphate chain lies nearly parallel to the adenine moiety. Mn2+ bound to a single, high-affinity site on the ATPase lies above and in the plane of the adenine ring. The distances from enzyme-bound Mn2+ to N6 and N7 are too large for first coordination sphere complexes, but are appropriate for second-sphere complexes involving, for example, intervening hydrogen-bonded water molecules. The NMR data also indicate that the structure of the bound ATP analogue is independent of the conformational state of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)