Aromatic 19F-13C TROSY - [19F, 13C] pyrimidine labeling for NMR spectroscopy of RNA.
Related Articles Aromatic 19F-13C TROSY - [19F, 13C] pyrimidine labeling for NMR spectroscopy of RNA.
Angew Chem Int Ed Engl. 2020 Jun 18;:
Authors: Kreutz C, Nußbaumer F, Plangger R, Roeck M
Abstract
We present the access to [5-19F, 5-13C] uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-19F, 5-13C]-pyrimidine labels into five RNAs - the 30nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61nt human hepatitis B virus ? (hHBV ?) RNA, the 49nt SAM VI riboswitch aptamer domain from B. angulatum, the 29nt apical stem loop of the pre-microRNA (miR) 21 and the 59nt full length pre-miR 21. The main stimulus to introduce the aromatic 19F-13C-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 13C-19F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-19F, 5-13C]/[5-19F] pyrimidine labeling. For the 61nt hHBV ? we find a beneficial 19F-13C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19F, 13C]-labeling of the SAM VI aptamer domain and the pre-miR 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.
PMID: 32558232 [PubMed - as supplied by publisher]
More...