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Aromatic 19 F– 13 C TROSY—[ 19 F, 13 C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA
Author(s) -
Nußbaumer Felix,
Plangger Raphael,
Roeck Manuel,
Kreutz Christoph
Publication year - 2020
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.202006577
Subject(s) - chemistry , rna , nuclear magnetic resonance spectroscopy , aptamer , pyrimidine , nucleotide , stereochemistry , biochemistry , microbiology and biotechnology , biology , gene
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 ϵ (hHBV ϵ ) 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 ϵ 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.