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.
Selective stable isotope labeling has transformed structural and dynamics analysis of RNA by NMR spectroscopy. These methods can remove13C-13C dipolar couplings that complicate13C relaxation analyses. While these phenomena are well documented for sites with adjacent13C nuclei (e.g. ribose C1′), less is known about so-called isolated sites (e.g. adenosine C2). To investigate and quantify the effects of long-range (> 2 Å)13C-13C dipolar interactions on RNA dynamics, we simulated adenosine C2 relaxation rates in uniformly [U-13C/15N]-ATP or selectively [2-13C]-ATP labeled RNAs. Our simulations predict non-negligible13C-13C dipolar contributions from adenosine C4, C5, and C6 to C2 longitudinal (R1) relaxation rates in [U-13C/15N]-ATP labeled RNAs. Moreover, these contributions increase at higher magnetic fields and molecular weights to introduce discrepancies that exceed 50%. This will become increasingly important at GHz fields. Experimental R1measurements in the 61 nucleotide human hepatitis B virus encapsidation signal ε RNA labeled with [U-13C/15N]-ATP or [2-13C]-ATP corroborate these simulations. Thus, in the absence of selectively labeled samples, long-range13C-13C dipolar contributions must be explicitly taken into account when interpreting adenosine C2 R1rates in terms of motional models for large RNAs.
more » « less- Award ID(s):
- 1808705
- NSF-PAR ID:
- 10224164
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Journal of Biomolecular NMR
- ISSN:
- 0925-2738
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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