Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2‐19F, 2‐13C]‐adenosine, which allow the incorporation of aromatic19F‐13C spin pairs. The labels when coupled with the transverse relaxation optimized spectroscopy (TROSY) enable us to probe RNAs comprising up to 124 nucleotides. With our new [2‐19F, 2‐13C]‐adenosine‐phosphoramidite, all resonances of the human hepatitis B virus epsilon RNA could be readily assigned. With [2‐19F, 2‐13C]‐adenosine triphosphate, the 124 nt pre‐miR‐17‐NPSL1‐RNA was produced via in vitro transcription and the TROSY spectrum of this 40 kDa [2‐19F, 2‐13C]‐A‐labeled RNA featured sharper resonances than the [2‐1H, 2‐13C]‐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 [2‐19F, 2‐13C]‐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 [2‐19F, 2‐13C]‐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.
A combined enzymatic and chemical synthesis of a 2′‐
- NSF-PAR ID:
- 10381137
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Current Protocols
- Volume:
- 2
- Issue:
- 7
- ISSN:
- 2691-1299
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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