We report on the determination of the solution structure of two sequence-related oligonucleotides, d(GTACGTAC)2 and d(CATCGATG)2. Results have been obtained by using a combined approach of (a) two-dimensional NMR, including proton and phosphorus experiments, (b) restrained molecular mechanics performed with sugar phase angle, backbone epsilon angle, and NOE distances as input, and (c) back-calculation refinements against the NOE spectra at various mixing times. The two oligonucleotides adopt the B-DNA structure with, however, noticeable differences centered on their core sequence and especially the CpG step. Due to the permutation of its flanking residues, the CpG step modifies its twist values and backbone epsilon value; globally, the CpG step appears more flexible within the tetranucleotide TCGA than ACGT. The solution structure of d(GTACGTAC)2 differs from the previously reported X-ray structure, which was found to be A-form throughout [Takusagawa, F. (1990) J. Biomol. NMR 3, 547-568]. On the other hand, in the X-ray structure of d(CCAACGTTGG)2 [Privé et al. (1991) J. Mol. Biol. 217, 177-199] the structure of the ACGT sequence is similar to that found in solution d(GTACGTAC)2. Similarly, the central TCGA tetranucleotide of d(CATCGATG)2 presents a solution structure analogous to that observed on the X-ray structures of both d(CGATCGATCG)2 [Grzeskowiak, et al. (1991) J. Biol. Chem. 266, 8861-8883] and d(CGATCGmeATCG)2 [Baïkalov, et al. (1993) J. Mol. Biol. 231, 768-784]. At the end we discuss the possible biological significance of the particular structures exhibited by the ACGT and TCGA tetranucleotides.

We report the analysis of the solution structure of the DNA duplex d(CTTCGAAG)2 compared to that of d(CATCGATG)2, the two oligonucleotides being related by the permutation of residues 2 and 7. An earlier study has demonstrated the malleability of CpG in the tetrad TCGA of d(CATCGATG)2 [Lefebvre et al. (1995) Biochemistry 34, 12019-12028]. Conformations of d(CTTCGAAG)2 were evaluated by (a) two-dimensional NMR, including proton and phosphorus experiments, (b) adiabatic mapping of the conformational space, (c) restrained molecular mechanics undertaken with sugar phase angle, epsilon-zeta difference angle, and NOE distances as input, and (d) back-calculation-refinement against NOE spectra at various mixing times. d(CTTCGAAG)2 like d(CATCGATG)2 exhibits a B-DNA conformation. However, significant differences are noted between the two oligonucleotides, extending up to the central CpG step, although this step resides in the same TCGA tetrad in both sequences. In structures obtained with refined NMR data, CpG adopts, for instance, a greater twist and a higher guanine phase within d(CTTCGAAG)2 compared to d(CATCGATG)2. In the former oligonucleotide, the structure of CpG resembles strikingly that found in the ACGT tetrad of the cAMP responsive element [Mauffret et al. (1992) J. Mol. Biol. 227, 852-875]. Moreover, two conformers with CpG either in the BII state (epsilon, zeta = g-, t) or in the BI state (epsilon, zeta = t, g-) are found equally stable for d(CTTCGAAG)2. The energy barrier from BI to BII comes to only 5.7 kcal/mol, and the path of the transition is very short. When calculations on d(CTTCGAAG)2 are performed taking the BI/BII equilibrium into account, the agreement with both the 1H and 31P data is found better than in the case with a single conformation taken alone. The BI/BII equilibrium may also occur in d(CATCGATG)2, but the amount of BII conformer is now found weaker compared to its analogue. The ability of the CpG phosphate groups to adopt the BII conformation could provide a satisfying explanation for the high mutation rates observed at these sites.