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

ABSTRACT: We present the access to [5-19 F, 5-13 C]-uridine and -cytidine phosphoramidites for the production of site-specifically modified RNAs up to 65 nucleotides (nts). The amidites were used to introduce [5-19 F, 5-13 C]-pyrimidine labels into five RNAs-the 30?nt human immunodeficiency virus trans activation response (HIV TAR) 2 RNA, the 61?nt human hepatitis?B virus ? (hHBV ?) RNA, the 49?nt SAM VI riboswitch aptamer domain from B. 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-13 C-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-19 F labeling scheme is now transferred to RNA. We provide a protocol for the resonance assignment by solid phase synthesis based on diluted [5-19 F, 5-13 C]/[5-19 F] pyrimidine labeling. For the 61?nt hHBV ? we find a beneficial 19 F-13 C TROSY enhancement, which should be even more pronounced in larger RNAs and will facilitate the NMR studies of larger RNAs. The [19 F, 13 C]-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.

SUBMITTER: Nußbaumer F 

PROVIDER: S-EPMC7540360 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature