In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA . TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by H-1, C-13, and N-15 NMR relaxation measurements on a DNA decamer d(CATTTGCATC).d(GATGCAAATG). The DNA decamer was enriched with 15% C-13 and 98% N-15 isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA . TTG moiety. Analysis of N-15 relaxation showed that the order parameter, S-2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer, The strong temperature dependence of guanosine NH S-2 in the CAA . TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA . TTG sequence were broadened in 2D H-1 NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the H-1 T-2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG . CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA . TTG sequence but extending over the CAAAT . ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the H-1, C-13, and N-15 T-1 rho values. H-1 and C-13 T-1 rho values showed a pulse field strength dependence, but N-15 T-1 rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 mus was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.
