The hairpin-to-coil equilibrium of the hexadecadeoxynucleotide d(ATCCTATTTTTAGGAT) was extensively studied by means of NMR, T-jump and UV. The thermodynamic and kinetic parameters for this equilibrium were determined, yielding a consistent picture of the dynamical behavior of this hairpin structure, which is shown to be a clear example of a situation in which the linebroadening of the imino proton resonances is not determined by the lifetime of the double helix. A comparative study of the homologous hairpins in which the size of the loop was elongated from 4 to 7 thymidine residues shows a monotonous decrease in Tm for the hairpin-to-coil transitions. This finding is in contrast with the view that the stability of hairpins reaches a maximum with a loop size of 6-7 residues. The NMR results indicate that the accessibility of the thymine bases in the loop towards solvent molecules or complementary nucleotides greatly depends on the size of the loop.

No abstract

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H 2 0 solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G' . C7' and C 2 . G'' should be reversed).The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.Nuclear magnetic resonance spectroscopy has emerged as one of the most important tools for the structure elucidation of biomacromolecules in solution and for the study of their conformations and conformational dynamics [l]. For a time the full exploitation of the potential of the technique was severely impaired owing to difficulties in assigning individual resonances in the spectra to nuclei located at different sites in the molecules. In the last five years this situation has changed dramatically as a result of the application of the nuclear Overhauser effect (NOE) to biomacromolecules [2] and the development and application of two-dimensional Fourier transform techniques [3 -71.In NMR studies of tRNA the imino proton resonances have played a major role. These resonances are relatively well resolved and contain information about the helical structure and dynamics of base pairs in the molecule. Using one-dimensional (1 D) NOE techniques a complete spectral assignment of nearly all imino protons of tRNAPh" from yeast has been achieved [8 -111, while the imino-proton spectra of some other tRNAs have partly been characterized [12, 131. Notwithstanding these accomplishments two-dimensional NOE spectroscopy is, in principle, a more powerful and more efficient method for the detection of NOES between neighbouring protons in biological macromolecules [5 -7, 14-161 since the method can elucidate complete networks of spin connectivities in a single experiment. At this point, however, it should be realized that the vast majority of 2D NOE experiments reported up till now involve relatively small biomolecules (relative molecular mass less than lOOOO), of which the non-exchangeable protons (observable in D 2 0 ) are most easily examined. If water-exchangeable protons, e.g. the imino protons in nucleic acids, are also to be studied, an additional problem has to be overcome owing to the technical necessity of suppressing the huge resonance of water protons Previously we have reported that 2D NOE spectroscopy is indeed feasible for a molecule of the size of tRNA even when the aforementioned problems of large dynamic range are involved [16, 171. In order to take full advantage of th...