A-DNA Octamers Research Articles

Structure of the A-DNA octamer d(GGCATGCC).

The crystal structure of the self-complementary DNA octamer d(GGCATGCC) has been determined to a resolution of 2.38 A and R factor of 17.1%. The structure is an A-DNA octamer duplex with Watson-Crick base pairing along its length. This sequence extends the investigation of octamer duplexes of the type d(GGX(4)CC) which assume P6(1) packing within the crystallographic unit cell. The A-DNA duplex is non-linear with a helix axis curvature of 7 degrees and bending towards the DNA minor groove. In view of the biological importance of protein recognition of some A-DNA sequences the minor-groove geometry and helix-axis curvature for this structure is compared with all others of the type d(GGXATXCC).

Structure of d(CCCTAGGG): comparison with nine isomorphous octamer sequences reveals four distinct patterns of sequence-dependent intermolecular interactions.

The self-complementary deoxyoctanuceotide d(CCCTAGGG) crystallizes as an A-type double helix in the space group P4(3)2(1)2, a = b = 42.22 and c = 24.90 A, with one strand per asymmetric unit. Using 1533 unique reflections at 1.9 A and I > 2sigma, the structure was solved by molecular placement and refined to a final R value of 16.4%. This structure is isomorphous with nine other tetragonal A-DNA octamers, possessing a central pyrimidine/purine step that is fully extended along the backbone with trans, trans conformations around the C4'-C5' and O5'-P bonds. A structural water, sandwiched between the pi-cloud of the terminal guanine and the N3 atom of G7 in the adjacent duplex, stabilizes an intermolecular base triplet with one hydrogen bond between the terminal cytosine and G6 of an adjacent duplex. Comparative analysis of this structure with the isomorphous A-DNA octamers reveals the importance of base sequence and minor-groove hydration in intermolecular interactions. The minor grooves, which provide both hydrophobic and polar interactions, allow for four patterns of sequence-dependent binding involving interduplex base triplets in which the third base is bonded through a single hydrogen bond. A conserved water molecule appears to be crucial in the stabilization of these intermolecular interactions, which resemble specific recognition motifs found in the crystal structures of the TATA-box/TBP protein complex.

The High Resolution Crystal Structure of the DNA Decamer d(AGGCATGCCT)

The crystal structure of the DNA decamer d(AGGCATGCCT) has been determined to a resolution of 1.3 Å and Rfactor of 13.9%. The structure has a unique conformation with each of the decamer single strands forming base-pairing interactions with two symmetry-related strands. The central eight bases of the decamer form an A-DNA octamer duplex with one symmetry-related strand whilst the terminal 5′ -Aand T-3′ bases are flipped out and away from the octamer helix axis to form base-pairing interactions with a second symmetry-related strand. These A·T base-pairs lie perpendicular to the crystallographic caxis and pack within the unit cell in conjunction with a symmetry-related A·T base-pair displaced by 3.4 Å along the caxis. A novel base triplet interaction of the type A∗(G·C) is present in the structure with interaction from the major groove side of the terminal 5′-A base to the minor groove of the central A-DNA octamer. This structure reports the first example of cobalt hexammine binding to a right-handed DNA duplex. The crystallographic asymmetric unit contains two cobalt hexammine ligands with one site in the major groove coordinating viahydrogen bonds to the 5′-AGG bases, and the second site located between DNA molecules and interacting with the oxygen atoms of phosphate groups.

Crystal Packing Effects on A-DNA Helix Parameters: A Comparative Study of the Isoforms of the Tetragonal & Hexagonal Family of Octamers with Differing Base Sequences

The helix and base pair parameters of A-DNA octamers have been compared having different base sequences both in the tetragonal and hexagonal crystal systems. For the eight structures in the tetragonal family, the twist, rise, slide, inclination and tilt are essentially the same, influenced by the similarity in crystal packing. The propeller twist and the base pair buckle display small sequence dependent variations. But the base pair roll appears to be changed by the specific intermolecular hydrogen bonding interactions. For four of the five octamer structures in the hexagonal family, the base pair rise, slide, inclination, tilt, as well as the propeller twist and buckle are all very similar, while the twist angle and the base pair roll are not. The intermolecular hydrogen bonding interactions seem to be primarily responsible for the differences in the roll angle in the tetragonal structures but for both the roll and twist angles in the hexagonal structures. These results demonstrate that the majority of the observed helix base pair parameters for A-DNA octamers in crystals are affected by the crystal packing environment, while a few parameters, like propeller twist and base pair buckle display some base sequence dependence.

Crystal and molecular structure of the A-DNA dodecamer d(CCGTACGTACGG) : Choice of fragment helical axis

The crystal structure of the dodecamer d(CCGTACGTACGG) has been determined at 2.5 å resolution. The crystals grow in the hexagonal space group P6 122, a = b = 46·2 A ̊ , c = 71·5 A ̊ with one strand as the asymmetric unit. Diffraction data were collected by the oscillation film method yielding 1664 unique reflections with an Rmerge of 0·04. The structure was solved by real-space rotational translational searches with idealized helical models of A, B and Z-DNA. The best agreement was given by an A-DNA model with its dyad axis along the diagonal crystallographic dyad axis, with an R-factor 0·43 and correlation coefficient of 0·59 for data between 10 and 5 Å. Iterative map fitting and restrained least-squares refinement and addition of 40 solvent molecules brought the R-factor to 0·15 and the correlation coefficient to 0·97 for all data between 8·0 and 2·5 Å. The stereochemistry of the atomic model is good, with a root-mean-square deviation in bond distances of 0·006 Å. This is the first example of an A-DNA containing a full helical turn. The dodecamer displays a novel packing motif. In addition to the characteristic contacts between the terminal base-pairs and the minor grooves of symmetry-related molecules, there are also minor groove to minor groove interactions not previously observed. The packing leaves an approximately 25 Å diameter solvent channel around the origin, along the c-axis. The presence of a prominent 3·4 Å meridional reflection and other diffuse features in the diffraction pattern provided evidence for the presence of disordered B-DNA along the c-axis, which can be accommodated in these solvent channels. The molecular conformation of the dodecamer also displays novel features. The dyad-related halves of the molecule are bent at an angle of 20 °, and the helical parameters are affected by this bend. Unlike the shorter A-DNA octamers, the dimensions of the major groove can be directly measured. Novel correlations between local helical parameters and global conformational features are presented. Most of the solvent molecules are associated with the major groove and the sugar-phosphate backbone.

Hexagonal crystal structure of the A-DNA octamer d(GTGTACAC) and its comparison with the tetragonal structure: correlated variations in helical parameters

The alternating DNA octamer d(GTGTACAC) has been grown in a novel hexagonal crystal form. The structure has been determined and refined to a 2-A resolution, with 51 water molecules. The A-DNA conformation is a variant of that observed for the tetragonal form of the same sequence (Jain et al., 1989) containing a bound spermine. The crystals belong to the space group P6(1)22, a = b = 32.40 A and c = 79.25 A, with one strand in the asymmetric unit. The new hexagonal structure was solved by rotation and translation searches in direct space and refined to a final R value of 12.7% by using 1561 unique reflections greater than 1.5 sigma (I). The electron density clearly shows that the penultimate A7 sugar had flipped into the alternative C2'-endo pucker. This dent in the molecule can be attributed to close intermolecular contacts. In contrast, in the tetragonal structure, the DNA is distorted in the central TA step, where the A5 backbone bonds C4'-C5' and O5'-P assume trans conformations. The hexagonal double helix more closely resembles the fiber diffraction A-DNA, compared to the tetragonal form. For instance, the tilt angle is higher (16 degrees vs 10 degrees), which is correlated with a larger displacement from the helix axis (3.5 vs 3.3), a lower rise per residue (2.9 vs 3.2), and a smaller major-groove width (6.1 vs 8.7), thus indicating that the variations in these global helical parameters are correlated. The propeller twist angles in both forms are higher for the G-C base pairs (15.3 degrees, 12.14 degrees) than for the A-T base pairs (10.8 degrees, 9.1 degrees), which is the reverse of the expected order. Unlike the tetragonal structure, the hexagonal crystal structure interestingly does not contain a bound spermine molecule. Our analysis reveals that the conformational differences between the tetragonal and hexagonal forms are not entirely due to the spermine binding, and crystal packing seems to play an important role.

Effect of Crystal Packing Environment on Conformation of the DNA Duplex: Molecular Structure of the A-DNA Octamer d(G-T-G-T-A-C-A-C) in Two Crystal Forms

The structure of the octamer d(G-T-G-T-A-C-A-C) was determined in two different crystal forms, tetragonal P43212 and hexagonal P6122. Although in both forms the octamer adopts an A-DNA structure, there are significant conformational differences between them. In particular, the P-05′ and the C5′-C4′ bonds of the middle adenine (A5) residue exhibit a distorted trans-trans conformation in the tetragonal form, while they adopt the standard gauche-, gauche+ conformation in the hexagonal form. These differences can be correlated with certain features of the crystal packing interactions in the two forms. Furthermore, a comparison of the structures of various A-DNA octamers reveals that the A-form can be divided into two subclasses such that the hexagonal structures have helical and base pair parameters that fall closer to fiber A-DNA values, while in the tetragonal structures these parameters deviate more from fiber A-DNA. These results indicate that environment plays a major role in determining DNA conformation.

Molecular structure of an A-DNA decamer d(ACCGGCCGGT).

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.

The Effect of Crystal Packing on Oligonucleotide Double Helix Structure

One of the questions that constantly is asked regarding x-ray crystal structure analyses of macromolecules is: To what extent is the observed crystal structure representative of the molecular conformation when free in solution, and to what degree is the structure perturbed by intermolecular crystal forces? This can be assessed with DNA oligomers because of an unusual aspect of crystallization self-complementary oligomers should possess a twofold symmetry axis normal to their helix axis, yet more often than not crystal of such oligomers do not use this internal symmetry. The two ends of the helix are crystallographically distinct though chemically identical. Complexes of DNA oligomers with intercalating drugs such as triostin A tend to use their twofold symmetry when they crystallize, whereas complexes with non-intercalating, groove-binding drugs ignore this symmetry unless the drug molecule is very small. A detailed examination of crystal packing in the dodecamer C-G-C-G-A-A-T-T-C-G-C-G provides an explanation of all of the foregoing behavior in terms of the mechanism of nucleation of DNA or DNA-drug complexes on the surface of a growing crystal. Asymmetry of the ends of the DNA helix is the price that is paid for efficient lateral packing of helices within the crystal. The actual end-for-end variation in standard helix parameters is compared with the experimental noise level as gauged by independent re-refinement of the same oligonucleotide structure where available, and with the observed extent of variation of these same parameters along the helix. Oligomers analyzed are the B-DNA dodecamer C-G-C-G-A-A-T-T-C-G-C-G, the A-DNA octamer G-G-T-A-T-A-C-C, and the phosphorothioate analogue of the B-DNA hexamer G-C-G-C-G-C. End-for-end variation, presumably the result of crystal packing is typically double the experimental noise level, and half the variation in the same parameter along the helix. Analysis of crystal packing in the phosphorothioate hexamer, which uses the same P212121 space group as the dodecamer, shows that the highly unsymmetrical B1 vs. BII backbone conformation probably is to be ascribed to crystal packing forces, and not to the sequence of the hexamer.

Ordered water structure in an A-DNA octamer at 1.7 A resolution.

The crystal structure of the deoxyoctamer d(G-G-Br U-A-BrU-A-C-C) was refined to a resolution of 1.7 A using combined diffractometer and synchrotron data. The analysis was carried out independently in two laboratories using different procedures. Although the final results are identical the comparison of the two approaches highlights potential problems in the refinement of oligonucleotides when only limited data are available. As part of the analysis the positions of 84 solvent molecules in the asymmetric unit were established. The DNA molecule is highly solvated, particularly the phosphate-sugar back-bone and the functional groups of the bases. The major groove contains, in the central BrU-A-BrU-A region, a ribbon of water molecules forming closed pentagons with shared edges. These water molecules are linked to the base O and N atoms and to the solvent chains connecting the O-1 phosphate oxygen atoms on each strand. The minor groove is also extensively hydrated with a continuous network in the central region and other networks at each end. The pattern of hydration is briefly compared with that observed in the structure of a B-dodecamer.