Enhanced TROSY Effect in [2-19 F, 2-13 C] Adenosine and ATP Analogs Facilitates NMR Spectroscopy of Very Large Biological RNAs in Solution.
Angew Chem Int Ed Engl
; 63(9): e202316273, 2024 Feb 26.
Article
em En
| MEDLINE
| ID: mdl-38185473
ABSTRACT
Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2-19 F, 2-13 C]-adenosine, which allow the incorporation of aromatic 19 F-13 C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124â
nucleotides. With our new [2-19 F, 2-13 C]-adenosine-phosphoramidite, all resonances of the human hepatitisâ
B virus epsilon RNA could be readily assigned. With [2-19 F, 2-13 C]-adenosine triphosphate, the 124â
nt pre-miR-17-NPSL1-RNA was produced via in vitro transcription and the TROSY spectrum of this 40â
kDa [2-19 F, 2-13 C]-A-labeled RNA featured sharper resonances than the [2-1 H, 2-13 C]-A sample. The mutual cancelation of the chemical-shift-anisotropy and the dipole-dipole-components of TROSY-resonances leads to narrow linewidths over a wide range of molecular weights. With the synthesis of a non-hydrolysable [2-19 F, 2-13 C]-adenosine-triphosphate, we facilitate the probing of co-factor binding in kinase complexes and NMR-based inhibitor binding studies in such systems. Our labels allow a straightforward assignment for larger RNAs via a divide-and-conquer/mutational approach. The new [2-19 F, 2-13 C]-adenosine precursors are a valuable addition to the RNA NMR toolbox and will allow the study of large RNAs/RNA protein complexes in vitro and in cells.
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RNA
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Adenosina
Idioma:
En
Ano de publicação:
2024
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Article