Self-complementary DNA Duplex Research Articles

Validation of the nearest-neighbor model for Watson-Crick self-complementary DNA duplexes in molecular crowding condition.

Recent advancement in nucleic acid techniques inside cells demands the knowledge of the stability of nucleic acid structures in molecular crowding. The nearest-neighbor model has been successfully used to predict thermodynamic parameters for the formation of nucleic acid duplexes, with significant accuracy in a dilute solution. However, knowledge about the applicability of the model in molecular crowding is still limited. To determine and predict the stabilities of DNA duplexes in a cell-like crowded environment, we systematically investigated the validity of the nearest-neighbor model for Watson–Crick self-complementary DNA duplexes in molecular crowding. The thermodynamic parameters for the duplex formation were measured in the presence of 40 wt% poly(ethylene glycol)200 for different self-complementary DNA oligonucleotides consisting of identical nearest-neighbors in a physiological buffer containing 0.1 M NaCl. The thermodynamic parameters as well as the melting temperatures (Tm) obtained from the UV melting studies revealed similar values for the oligonucleotides having identical nearest-neighbors, suggesting the validity of the nearest-neighbor model in the crowding condition. Linear relationships between the measured ΔG°37 and Tm in crowding condition and those predicted in dilute solutions allowed us to predict ΔG°37, Tm and nearest-neighbor parameters in molecular crowding using existing parameters in the dilute condition, which provides useful information about the thermostability of the self-complementary DNA duplexes in molecular crowding.

Synthesis and structure of a new mononuclear copper(II) complex with 2,2ʹ-bipyridine and picrate: molecular docking, DNA-binding, and in vitro anticancer activity

A new mononuclear copper(II) complex identified as [Cu(bpy)2(pic)](pic), where bpy represents 2,2ʹ-bipyridine and pic stands for picrate (pic), has been synthesized and characterized by elemental analysis, molar conductivity, IR and electronic spectral studies, and single-crystal X-ray diffraction. The crystal structure analysis reveals that copper(II) has a distorted square-pyramidal coordination geometry. The hydrogen bonding and π–π stacking interactions contribute to a 3-D supramolecular structure in the crystal. The DNA-binding properties of the copper(II) complex are investigated both theoretically and experimentally, revealing that the copper(II) complex interacts with herring sperm DNA (HS-DNA) by intercalation, and the molecular docking of the copper(II) complex with the self-complementary DNA duplex of sequence d(ACCGACGTCGGT)2 facilitates the binding events. The in vitro anticancer activities suggest that the copper(II) complex is active against selected tumor cell lines. The influence of the complexation of pic in the mononuclear copper(II) complexes on DNA-binding properties and in vitro anticancer activities is discussed.

Synthesis and crystal structure of a ternary copper(II) complex of 2,2′-bipyridine and picrate: Molecular docking, reactivity towards DNA and in vitro anticancer activity

A new mononuclear ternary copper(II) complex with mixed ligands of 2,2′-bipyridine (bpy) and picrate (pic), namely [Cu(bpy)(pic)2], has been synthesized and characterized by elemental analysis, molar conductivity measurement, IR and electronic spectral studies, and single-crystal X-ray diffraction. The crystal structure analysis reveals the presence of two crystallographic independent molecules in an asymmetric unit. The copper(II) atoms are in elongated octahedral coordination geometries. A three-dimensional supermolecular network is formed through non-classical C−H⋯O hydrogen bonds. The DNA-binding properties of the copper(II) complex are investigated both theoretically and experimentally, revealing that the copper(II) complex can interact with HS-DNA in the mode of intercalation, and the molecular docking of the copper(II) complex with the self-complementary DNA duplex of sequence d(ACCGACGTCGGT)2 facilitates the binding events. The in vitro anticancer activities suggest that the copper(II) complex is active against the selected tumor cell lines.

Syntheses and crystal structures of tetracopper(II) complexes bridged by asymmetric N,N′-bis(substituted)oxamides: Molecular docking, DNA-binding and in vitro anticancer activity

Two new tetranuclear copper(II) complexes of the formulae [Cu4(oxbm)2(phen)2](NO3)2⋅6H2O (1) and [Cu4(oxbpa)2(phen)2](ClO4)2·4H2O (2), where H3oxbm and H3oxbpa stand for N-(2-aminopropyl)-N′- (2-carboxylatophenyl)oxamide and N-hydroxypropyl-N′-(2-carboxylatophenyl)oxamide, respectively, and phen is 1,10-phenanthroline, have been synthesized and characterized by elemental analyses, molar conductivity measurements, IR and electronic spectrum studies, and X-ray single crystal diffraction. In the two tetracopper(II) complexes, the presence of the circular tetracopper(II) cations is assembled by a pair of cis-oxamido-bridged dicopper(II) units through carboxyl bridges, in which Cu1 is located in a distorted square-planar environment, while Cu2 is in a distorted square-pyramidal geometry. Numerous hydrogen bonds link complex 1 or 2 into a 2-D infinite network. The interactions of the two tetracopper(II) complexes with DNA are investigated both theoretically and experimentally, revealing that these tetracopper(II) complexes can interact with HS-DNA in the mode of intercalation, and complex 1 possesses stronger intercalating ability. The molecular docking of the two tetranuclear copper(II) complexes with the self-complementary DNA duplex of sequence d(ACCGACGTCGGT)2 facilitates the binding events. Cytotoxicity experiments indicate that the two tetracopper(II) complexes exhibit cytotoxic effects against human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549. Interestingly, the cytotoxic activities of the two tetracopper(II) complexes are consistent with their DNA-binding abilities, following the order of 1>2. The main results suggest that different bridging ligands in tetracopper(II) complexes may play an important role in the DNA-binding properties and cytotoxic activities.

Synthesis and crystal structure of a new copper(II) complex with N,N′-(4,4′-bithiazole-2,2′-diyl)diacetimidamide as ligand: Molecular docking, DNA-binding and cytotoxicity activity studies

A new mononuclear copper(II) complex with formula of [Cu2H(DABTA)2](pic)⋅6H2O, where H2DABTA and pic− stand for N,N′-(4,4′-bithiazole-2,2′-diyl)diacetimidamide and picrate ion, respectively, has been synthesized and characterized by elemental analysis, molar conductivity measurement, IR and electronic spectra studies, and single-crystal X-ray diffraction. The crystal structure reveals that the copper(II) ion has a {CuN4} square-planar coordination environment. The solvent water molecules form a column parallel to c axis by hydrogen bonds. Then the mononuclear copper complexes link to the water columns to make a three-dimensional hydrogen bonding grid with the cavities filled by pic− anions. Besides, there are offset π–π stacking interactions between thiazole rings in the supramolecular system. The interactions between the copper(II) complex and herring sperm DNA (HS-DNA) have been investigated by using electronic absorption titration, fluorescence titration and viscometry. The molecular docking of the complex with the self-complementary DNA duplex of sequence d(ACCGACGTCGGT)2 demonstrates that the complex is stabilized by additional electrostatic and hydrogen bonding interaction with the DNA. The copper(II) complex exhibits potent anticancer activities against human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549.

Structural and energetic characterization of nucleic acid-binding to the fingers domain of Moloney murine leukemia virus reverse transcriptase.

Reverse transcriptase is an essential retroviral enzyme that replicates the single-stranded RNA genome of the retrovirus producing a double-stranded DNA copy, which is subsequently integrated into the host's genome. We have previously reported that processive DNA synthesis of Moloney murine leukemia virus reverse transcriptase (MMLV RT) is severely compromised by substitution of an Ala for the fingers domain residue Arg 116. In order to further investigate the role of Arg 116 in interactions of MMLV RT with nucleic acids, we have determined the crystal structure of the R116A N-terminal fragment and characterized the binding of two self-complementary DNA duplexes [d(CATGCATG)2 and d(CGCGCGCG)2] to both the wild-type and R116A fragments by isothermal titration calorimetry. The resultant thermodynamic profiles extrapolated to 25 degrees C reveal that binding of the wild-type N-terminal fragment to both DNA duplexes is enthalpy-driven and characterized by an unfavorable entropy. Although the temperature dependence of the respective protein-DNA binding enthalpies is markedly different reflecting distinct heat capacity changes, the binding free energies are nearly identical and relatively invariant to temperature (DeltaG approximately -6.0 kcal x mol(-1)). In contrast to the wild-type fragment, the R116A fragment exhibits no measurable affinity for either DNA duplex, yet its crystal structure reveals no significant changes when compared to the wild-type structures. We suggest that hydrogen-bonding interactions involving the fingers domain residue Arg 116 are critical for DNA binding as well as processive DNA synthesis by MMLV RT.

Trans-hydrogen bond deuterium isotope effects of A:T base pairs in DNA.

The chemical shifts of (13)C2 of adenosine residues of DNA were observed to experience a through-space or trans-hydrogen bond isotope effect as a result of deuterium substitution at the imino hydrogen site of base-paired thymidine residues. NMR measurements of several self-complementary DNA duplexes at natural abundance (13)C in 50% H(2)O, 50% D(2)O solvent mixtures yielded an average trans-hydrogen bond isotope effect, (2h)Delta(13)C2, of -47 ppb. The data suggest that stronger hydrogen bonds have more negative (2h)Delta(13)C2 values, which means that A:T N1.H3 hydrogen bonds increase the anharmonicity of the effective vibrational potential of H3. However, (2h)Delta(13)C2 values do not correlate with intra-residue (2)Delta(13)C4 values of thymidine observed here and earlier (Vakonakis et al., 2003), which suggests that (2h)Delta(13)C2 is not determined entirely by hydrogen bond strength. Instead, the variations observed in (2h)Delta(13)C2 values suggest that they may also be sensitive to base pair geometry.

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 ∼ 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 ∼ 180° and ∼ 120°, respectively while all other residues are close to C2′endo-conformation. A large propeller twist (∼ −18°) and smallest twist angle (∼ 31°) at A3pT4 step, in the middle of the sequence, a wider (12 Å) and shallower (3.0 Å) 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.

Solution structure of the DNA complexes with an alkyl-linked bis(benzimidazole) compound, 1,4-bis[5-(4-methyl-1-piperazinyl)-2-benzimidazolyl]butane.

Solution structures of the 1:1 complexes of an alkyl-linked bis(benzimidazole) compound (4; Figure 1(a)) with the self-complementary DNA duplexes, d(CGCGAATTCGCG)2 and d(CGCAAATTTGCG)2, have been studied by 1H NMR spectroscopy and molecular mechanics calculations. The intermolecular NOE data and the energy-minimized geometries of the 1:1 complexes indicate that 4 binds in the minor groove of the 5'-GAATTCG and 5'-CAAATTT regions of the DNA duplexes.

DNA melting in the presence of fluorescent intercalating oxazole yellow dyes measured with a gel-based assay.

We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is approximately 2.5 degrees C for each intercalation site occupied in the presence of 38 mM [Na(+)], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied.

Solution structures of the DNA complexes with alkyl-linked bis(benzimidazole) compounds studied by NMR spectroscopy.

We have synthesized two novel alkyl-linked bis(benzimidazole) compounds related to pentamidine. The solution structures of 1:1 complexes of these compounds with the self-complementary DNA duplexes, d(CGCGAATTCGCG)2 (A2T2) and d(CGCAAATTTGCG)2 (A3T3) have been studied by one- and two-dimensional 1H NMR spectroscopy. The intermolecular NOE data of the A3T3 complexed with compound 3 indicate that 3 binds in the minor groove of the central 5'-CAAATTTG region of A3T3.

The First Example of a Hoogsteen Basepaired DNA Duplex in Dynamic Equilibrium with a Watson-Crick Basepaired Duplex—A Structural (NMR), Kinetic and Thermodynamic Study

A single-point substitution of the O4′ oxygen by a CH2 group at the sugar residue of A 6 (i.e. 2′-deoxyaristeromycin moiety) in a self-complementary DNA duplex, 5′- d(C1G2C3G4A5A6T7T8C9G10C11G12)2 −3, has been shown to steer the fully Watson-Crick basepaired DNA duplex (1A), akin to the native counterpart, to a doubly A 6:T7 Hoogsteen basepaired (1B) B-type DNA duplex, resulting in a dynamic equilibrium of (1A)→←(1B): Keq = k1/k-1 = 0.56±0.08. The dynamic conversion of the fully Watson-Crick basepaired (1A) to the partly Hoogsteen basepaired (1B) structure is marginally kinetically and thermodynamically disfavoured [k1 (298K) = 3.9± 0.8 sec−1; δH°‡ = 164±14 kJ/mol;-TδS°‡ (298K) = −92 kJ/mol giving a δG298°‡ of 72 kJ/mol. Ea (k1) = 167±14 kJ/mol] compared to the reverse conversion of the Hoogsteen (1B) to the Watson-Crick (1A) structure [k-1 (298K) = 7.0±0.6 sec-1, δH°‡ = 153±13 kJ/mol;-TδS°‡ (298K) = −82 kJ/mol giving a δG298°‡ of 71 kJ/mol. Ea (k-1) = 155±13 kJ/mol]. A comparison of δG298°‡ of the forward (k1) and backward (k-1) conversions, (1A)→←(1B), shows that there is ca 1 kJ/mol preference for the Watson-Crick (1A) over the double Hoogsteen basepaired (1B) DNA duplex, thus giving an equilibrium ratio of almost 2:1 in favour of the fully Watson-Crick basepaired duplex. The chemical environments of the two interconverting DNA duplexes are very different as evident from their widely separated sets of chemical shifts connected by temperature-dependent exchange peaks in the NOESY and ROESY spectra. The fully Watson-Crick basepaired structure (1A) is based on a total of 127 intra, 97 inter and 17 cross-strand distance constraints per strand, whereas the double A 6:T7 Hoogsteen basepaired (1B) structure is based on 114 intra, 92 inter and 15 cross-strand distance constraints, giving an average of 22 and 20 NOE distance constraints per residue and strand, respectively. In addition, 55 NMR-derived backbone dihedral constraints per strand were used for both structures. The main effect of the Hoogsteen basepairs in (1B) on the overall structure is a narrowing of the minor groove and a corresponding widening of the major groove. The Hoogsteen basepairing at the central A 6:T7 basepairs in (1B) has enforced a syn conformation on the glycosyl torsion of the 2′- deoxyaristeromycin moiety, A 6, as a result of substitution of the endocyclic 4′-oxygen in the natural sugar with a methylene group in A 6. A comparison of the Watson-Crick basepaired duplex (1A) to the Hoogsteen basepaired duplex (1B) shows that only a few changes, mainly in α, σ and γ torsions, in the sugar-phosphate backbone seem to be necessary to accommodate the Hoogsteen basepair.

Solution structure of the DNA complex with a quinacrine-netropsin hybrid molecule by NMR spectroscopy.

The solution structures of 1:1 complexes of a quinacrine-netropsin hybrid molecule with the self-complementary DNA duplexes, d(CGCGAATTCGCG)2 and d(CGAATTCG)2, have been studied by one- and two-dimensional 1H NMR spectroscopy. The NOE data indicate that the acridine ring of the hybrid intercalates into the 5'-GpA step and its netropsin moiety spans the minor groove of the central AATT region.

Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies.

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.

Factors Contributing to Aromatic Stacking in Water: Evaluation in the Context of DNA

We report the use of thermodynamic measurements in a self-complementary DNA duplex (5'-dXCGCGCG)(2), where X is an unpaired natural or nonnatural deoxynucleoside, to study the forces that stabilize aqueous aromatic stacking in the context of DNA. Thermal denaturation experiments show that the core duplex (lacking X) is formed with a free energy (37 °C) of -8.1 kcal·mol(-1) in a pH 7.0 buffer containing 1 M Na(+). We studied the effects of adding single dangling nucleosides (X) where the aromatic "base" is adenine, guanine, thymine, cytosine, pyrrole, benzene, 4-methylindole, 5-nitroindole, trimethylbenzene, difluorotoluene, naphthalene, phenanthrene, and pyrene. Adding these dangling residues is found to stabilize the duplex by an additional -0.8 to -3.4 kcal·mol(-1). At 5 μM DNA concentration, T(m) values range from 41.7 °C (core sequence) to 64.1 °C (with dangling pyrene residues). For the four natural bases, the order of stacking ability is A > G ≥ T = C. The nonpolar analogues stack more strongly in general than the more polar natural bases. The stacking geometry was confirmed in two cases (X = adenine and pyrene) by 2-D NOESY experiments. Also studied is the effect of ethanol cosolvent on the stacking of natural bases and pyrene. Stacking abilities were compared to calculated values for hydrophobicity, dipole moment, polarizability, and surface area. In general, hydrophobic effects are found to be larger than other effects stabilizing stacking (electrostatic effects, dispersion forces); however, the natural DNA bases are found to be less dependent on hydrophobic effects than are the more nonpolar compounds. The results also point out strategies for the design nucleoside analogues that stack considerably more strongly than the natural bases; such compounds may be useful in stabilizing designed DNA structures and complexes.

Crystal Structures of Oligodeoxyribonucleotides Containing 6‘-α-Methyl and 6‘-α-Hydroxy Carbocyclic Thymidines

The X-ray crystal structures of two self-complementary DNA duplexes with the sequence d(CGCGAAtMe/OHtMe/OHCGCG) containing four 6‘-α-methyl (tMe) or four 6‘-α-hydroxy carbocyclic thymidines (tOH), respectively, have been determined. Both structures are isomorphous to the native Dickerson−Drew dodecamer duplex [d(CGCGAATTCGCG)]2. The cyclopentane moieties of the modified carbocyclic thymidine residues lacking the deoxyribose 4‘-oxygen adopt either C2‘-endo or C1‘-exo B-DNA type puckers. The dodecamer duplex incorporating 6‘-α-methyl carbocyclic thymidines shows an enlarged minor groove relative to both the unmodified duplex and the one with incorporated 6‘-α-hydroxy carbocyclic thymidines. The pairing of oligonucleotides containing single or multiple 6‘-α-substituted carbocyclic thymidines with complementary DNA is discussed on the basis of the structural data.

Experimental Measurement of Aromatic Stacking Affinities in the Context of Duplex DNA

Experimental Measurement of Aromatic Stacking Affinities in the Context of Duplex DNA

Solution structure of the conserved segment of the Myb cognate DNA sequence by 2D NMR, spectral simulation, restrained energy minimization, and distance geometry calculations.

Solution structure of a self-complementary DNA duplex d-ACCGTTAACGGT containing the TAACGG recognition segment of Myb protein has been obtained by NMR spectroscopy. Complete resonance assignments of all the protons (except H5', H5" protons) have been obtained following standard procedures based on two-dimensional NMR techniques. Using a total of 72 coupling constants, and 95 NOE intensities, restrained energy minimization has been carried out, with the X-PLOR force field. The distance constraint set has been iteratively refined, for better fits with experimental NOE intensities. Using the final constraint set thus obtained, and explicit H-bond constraints for A.T, G.C base pairs in the duplex, distance geometry calculations have been carried in the torsion angle space with the program TANDY-2S to identify the family of structures consistent with the NMR data. We observe that the constraint set does indeed define a unique structure for the DNA segment. The structural details have been analyzed, and the sequence-dependent variations in torsion angles, base pair geometries, and helicoidal parameters have been documented. We observed that the helix axis displays a nonregular path, and three centered H-bonds have been seen at AA, AC, and CC steps in the major groove of the helix. Substantial variations have been observed for the helix axis and the groove widths at the recognition site. The base pairs exhibit high negative propeller twists. The structure is characterized by O4'-endo geometry for all the sugar rings (expect G10), and the other torsion angles belong to the B-DNA families.

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure

The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.

Solution structure of GCCAAT recognition motif by 2D NMR, spectral simulation, molecular modeling, and distance geometry calculations.

Solution conformation of a self-complementary 14-mer DNA duplex (d-GGATTGGCCAATCC) containing the GCCAAT recognition motif of several transcription factors has been investigated by NMR spectroscopy. Complete resonance assignment of all the protons (except H5',H5'' protons) has been obtained following standard procedures based on two-dimensional NMR techniques. Three-bond coupling constants have been determined by spectral simulation procedures. New strategies have been described and employed for quantifying NOE intensities from the structural point of view. Approximate ranges of gamma torsion angles have been obtained from a selective NOESY experiment, by estimating the J(4'-5'), J(4'-5''), or their sum in the H1'-H4' cross peaks of the spectrum. Likewise, ranges of epsilon torsion angles have been obtained by monitoring the H3' multiplicities in the H8/H6-H3' cross peaks in selective NOESY spectra. With the help of such a total of 73 coupling constraints, 79 NOE intensity constraints, and 108 H-bond constraints, model building has been carried out to obtain a structure which satisfies the constraints. Starting from such a structure, an expanded distance constraint set has been created which has been used for the distance geometry calculations using the program TANDY. In the best structure thus derived, interesting irregularities similar to a BI-BII transition have been observed in the center. The molecule exhibits a bend. The overall base stacking is different from that in either B- or A-DNA models. The base pairs are tilted with respect to the local helix axes. The observed structural features are likely to have important implications for the recognition mechanism of the GCCAAT motif.