Aromatic ¹⁹F–¹³C TROSY—[¹⁹F, ¹³C]‐Pyrimidine Labeling for NMR Spectroscopy of RNA

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 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 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 61 nt hHBV ϵ 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‐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.

A suite of 19F based relaxation dispersion experiments to assess biomolecular motions

“…In general, 19 F based experiment can thus be expected to outperform more traditional measurements at the elevated temperatures or at a higher pH. Finally, recent advances in labeling techniques have made RNA and DNA accessible for 19 F NMR (Sochor et al 2016;Nußbaumer et al 2020;Baranowski et al 2020), thereby further expanding the applicability of the presented methodologies. In summary, our approach will strengthen the use of 19 F NMR to accurately quantify dynamic processes in a broad range of biological systems.…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“…19 However since that time, Aryl 19 F- 13 C TROSY methods have been reported for biopolymers, 36 and have now been applied to ligand binding. 50 However, only a limited set of fluorinated amino acids and nucleic acids have been used, in part due to the requirement of specific 13 C and 19 F labeling. Improved access to these building blocks may augment the types of biopolymers which can be studied by this approach.…”

“… 36 This has led to the characterization of proteins as large as 180 kDa, and has more recently been applied to 13 C-labeled fluorouracil in RNA including ligand binding studies. 50 Conversely, CF 3 -TROSY may be limited to lower field strengths where the CSA effects are negligible. At high field strengths, TROSY effects were not observed due to the high CSA of the CF 3 group.…”

Synthesis of [7-15N]-GTPs for RNA structure and dynamics by NMR spectroscopy

“…Even with these innovations, spectral crowding and signal overlap still limit the effective analysis of large (> 30 nucleotide, nt) RNAs [18]. To tackle this problem, we and others have developed labeling technologies to incorporate stable isotopes (i.e., 2 H, 13 C, 19 F, and 15 N) into RNA to benefit NMR structure and dynamics measurements [7,[19][20][21][22][23][24][25][26][27].…”