Isotope-assisted structural chemical analysis of nucleic acids

Yoshiyuki Tanaka

Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba, Aramaki,
Aoba-ku, Sendai 980-8578, Japan
tanaka@mail.pharm.tohoku.ac.jp

In the mechanistic/physicochemical studies of biological molecules, it is important to probe catalytic sites and molecular interfaces. In order to probe a specific site in a biological macromolecule, site-specific isotope labeling is a straight-forward way. By using the site-specifically labeled RNA and DNA molecules, we have studied their structures and physicochemical properties. To date, with the site-specific labeling technique, we have studied 1) a weak hydrogen bond of a C-U mismatched base-pair in an RNA duplex, 2) metal cation-binding to conserved sequence motif of hammerhead ribozyme and 3) a chemical structure determination of Hg^II-mediated T-T base-pair (T-Hg^II-T base-pair) in a DNA duplex. In all systems, a single atom or a single residue was site-specifically labeled with ¹⁵N. For the detection of hydrogen-bonding and metal ion-binding, the traces of ¹⁵N chemical shifts against temperature (hydrogen bond) and metal cation-concentration (metal ion-binding) was efficient. For the determination of the chemical structure of the T-Hg^II-T base-pair, we detected the 2-bond ¹⁵N-¹⁵N J-coupling through the N-Hg^II-N bond (²J_NN). In addition, the direction of chemical shift perturbations can be qualitatively predictable for imine-type nitrogen, based on the theory of the chemical shift and the bonding state of the nitrogen atoms. Thus, NMR spectroscopic parameters of ¹⁵N nuclei are sensitive probe for micro-environment around the corresponding nitrogen atoms. Lastly, site-specific labeling technique for "long" RNA molecules will be presented.

This works was supported by Human Frontier Science Program.