Aromatic19F-13C TROSY-[19F,13C]-Pyrimidine Labeling for NMR Spectroscopy of RNA

We present the access to [5-F-19, 5-C-13]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-F-19, 5-C-13]-pyrimidine labels into five RNAs-the 30 nt human immunodeficiency virustransactivation response (HIV TAR) 2 RNA, the 61 nt human hepatitis B virus epsilon(hHBV epsilon) RNA, the 49 nt SAM VI riboswitch aptamer domain fromB. angulatum, the 29 nt apical stem loop of the pre-microRNA (miRNA) 21 and the 59 nt full length pre-miRNA 21. The main stimulus to introduce the aromatic(19)F-C-13-spin topology into RNA comes from a work of Boeszoermenyi et al., in which the dipole-dipole interaction and the chemical shift anisotropy relaxation mechanisms cancel each other leading to advantageous TROSY properties shown for aromatic protein sidechains. This aromatic(13)C-F-19 labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-F-19, 5-C-13]/[5-F-19] pyrimidine labeling. For the 61 nt hHBV epsilon we find a beneficial(19)F-C-13 TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [F-19,C-13]-labeling of the SAM VI aptamer domain and the pre-miRNA 21 further opens the possibility to use the biorthogonal stable isotope reporter nuclei in in vivo NMR to observe ligand binding and microRNA processing in a biological relevant setting.