Solution structure of the CpG containing d(CTTCGAAG)(2) oligonucleotide: NMR data and energy calculations are compatible with a BI/BII equilibrium at CpG

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 H-1 and P-31 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 Bn. conformation could provide a satisfying explanation for the high mutation rates observed at these sites.