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Discrimination of intra- and intermolecular hydrogen bonds in a symmetric multimer has not been accomplished yet, although such discrimination would provide a crucial basis for construction of the multimeric architecture of nucleic acids by NMR. We have developed a direct and unambiguous method for such discrimination involving the use of scalar couplings across hydrogen bonds. The method has been validated with a symmetric dimer of d(GGGCTTTTGGGC), for which the structure including both intra- and intermolecular hydrogen bonds was already reported. This has demonstrated that our method can clearly discriminate these two kinds of hydrogen bonds. Then, the method was applied to a symmetric dimer of d(GGAGGAGGAGGA) and has provided decisive information on its multimeric architecture. Additionally, the values for scalar couplings across hydrogen bonds for G:G and G:A base pairs in the G(:A):G(:A):G(:A):G heptad formed by d(GGAGGAGGAGGA) were determined for the first time. This determination has provided an insight into the nature of the heptad.

We have reported that d(GGAGGAA) forms a new type of quadruplex in the presence of K+. Here we report that in the absence of K+ it forms another kind of new structure composed of non-standard base pairs exclusively. The structure is neither a quadruplex nor a hairpin. The structure gives three clear imino proton signals of G at 10.0, 13.0 and 13.8 ppm, indicating the formation of G:A and/or G:G base pairs. The two signals at 13.0 and 13.8 ppm indicate that there are two base pairs in which the imino protons of G are hydrogen bonded. All residues take anti conformation, which imposes the restriction on the combination of a mutual direction of strands and types of base pairs. Phosphorus signals of the second and fifth residues appear at higher field, while that of the seventh residue at lower field, when compared with those of A and B form structures, suggesting deviations of a backbone conformation from the standard one. Replacement of the second or fifth G by I results in destabilization of the structure, which implies the involvement of amino groups of these residues in base pairing. On the basis of these results, the structure of d(GGAGGAA) is discussed.

The structures of two oligodeoxyribonucleotide duplexes, the base sequences of which were modelled after both a hammerhead ribozyme and a small metalloribozyme, were studied by NMR. Both duplexes contain adjacent G:A mismatches; one has a PyGAPu:PyGAPu sequence and the other a PyGAPy:PuGAPu sequence. It is concluded on the basis of many characteristic NOEs that in both duplexes G:A base pairs are formed in the unique 'sheared' form, where an amino proton instead of an imino proton of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head'. A photo-CIDNP experiment, which gives unique and independent information on the solvent accessibility of nucleotide bases, also supports G:A base pairing rather than a bulged-out structure of G and A residues. This is the first demonstration that not only the PyGAPu:PyGAPu sequence but also the PyGAPy:PuGAPu sequence can form the unique sheared G:A base pairs. Taking the previous studies on G:A mismatches into account, the idea is suggested that a PyGA:GAPu sequence is a minimum and essential element for the formation of the sheared G:A base pairs. The sheared G:A base pairs in the PyGAPu:PyGAPu sequence are suggested to be more stable than those in the PyGAPy:PuGAPu sequence. This is explained rationally by the idea proposed above.

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.

The conformations of double-stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) and d(GCATTTTGAAACG)d(CGTTTCAAAATGC) have been studied by NMR spectroscopy. Analyses of cross peaks in NOESY spectra between the H2 of an adenine and the H1' of a deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' in each duplex. In view of this gradual compression of the minor groove along the oligo(dA) tract, it can be understood clearly why d(GGAAATTTCC)n x 2 and d(GAAAATTTTC)n x 2 are bent, and d(GGTTTAAACC)n x 2 and d(GTTTTAAAAC)n x 2 are not bent. The relative extents of bending of a series of d(AjN10-j)nd(N10-jTj)n sequences can also be understood systematically. Additionally, it was found that the TA step disturbed the compression of the minor groove of the oligo(dA) tract to some extent.

Eighteen kinds of oligodeoxyribonucleic acids have been examined to reveal their structures in aqueous solutions at different ionic strengths by Raman spectroscopy. The structures in solutions were found to be very polymorphic depending on their sequences as well as on the salt concentrations. At a low salt condition a DNA oligomer assumes a unique B form within a B family, for examples Ba, Bh, B', or Bn form. Amongst these DNA oligomers, d(CGCG)2 showed a salt induced Ba-Z transition, while d(GGGGCCCC)2 showed a salt induced Bh-A transition. DNA oligomers with AA/TT sequences were found to prefer B' form even at high salt condition. From comparing the structures of DNA oligomers in solutions with their crystal structures, it is safe to say that the crystal structure of a DNA oligomer is very similar to the structure in the high salt solution.

The resonances of the imino protons and all of the non-exchangeable protons (except for H5'/H5'') of d(CGCAAAAAAGCG)d(CGCTTTTTTGCG) have been assigned by means of one- and two-dimensional NMR spectroscopies. Qualitative analyses showed that the overall structure is of the B-form, but local conformational deviations exist. The NOEs between the imino protons of thymines and H2 of adenines suggest that the A-T base pairs are propeller-twisted to almost the same degree as in crystals. A remarkable chemical shift of H1' was observed for the residue located just before the oligo(dA)oligo(dT) tract, suggesting the presence of conformational discontinuity at the junctions between the oligo(dA)oligo(dT) tract and the other portions. Analyses of cross peaks in NOESY spectra between H2 of adenines and H1' of the 3'-neighbouring residues on the complementary strand revealed that the minor groove of the oligo(dA)oligo(dT) tract is narrow and compressed gradually, from 5' to 3', along the tract.

The conformations of double stranded d(GGAAATTTCC) x 2, d(GGTTTAAACC) x 2, d(CGCAAAAAAGCG).d(CGCTTTTTTGCG) and d(GCATTTTGAAACG).d(CGTTTCAAAATGC) have been studied by means of NMR spectroscopy. Analyses of cross peaks in NOESY spectra between H2 of an adenine and H1' of the deoxyribose in the 3'-neighbouring residue on the complementary strand revealed that the minor groove of the oligo(dA) tract is compressed gradually from 5' to 3' along the tract in four oligonucleotides. A new model is proposed as to DNA bending based on the evidence of gradual compression of the minor groove. This model can explain why d(GGAAATTTCC) n x 2 and d(GAAAATTTTC) n x 2 are bent, and d(GGTTTAAACC) n x 2 and d(GTTTTAAAAC) n x 2 are not bent. The bending of d(AjN10-j) n x 2 sequences can also be explained.