Comparing Strong and Weak Halogen Bonding in Solution: 13 C NMR, UV/Vis, Crystallographic, and Computational Studies of an Intramolecular Model: Comparing Strong and Weak Halogen Bonding in Solution: 13 C NMR, UV/Vis, Crystallographic, and Computational Studies of an Intramolecular Model

Widner, Danielle L.; Robinson, Emily R.; Perez, Alejandra B.; Vang, Herh G.; Thorson, Rachel A.; Driscoll, Zakarias L.; Giebel, Sierra M.; Berndt, Calvin W.; Bosch, Eric; Speetzen, Erin D.; Bowling, Nathan P.  (ORCID:0000000269301333)

doi:10.1002/ejoc.201700969

Abstract

Large RNAs are central to cellular functions, but characterizing such RNAs remains challenging by solution NMR. We present two labeling technologies based on [2‐¹⁹F, 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.

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