³¹P nmr spin‐lattice relaxation studies of deoxyoligonucleotides

Temperature‐dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin‐lattice relaxation times (T₁), and ³¹P‐{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′‐5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double‐ and single‐stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10⁻¹⁰ sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double‐helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P‐O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ≅ 10⁻⁹ sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10⁻¹⁰ sec, even for dinucleotides.