Evaluation of 15N-detected H–N correlation experiments on increasingly large RNAs
Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15N-detected H–N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-detected H–N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.
Keywords15N direct detection RNA BEST-TROSY Line width analysis Field dependence Exchange
Matthias Görlach from the Core Facilities and Services (CS Protein Production) of the Fritz Lipmann Institute, Jena, is gratefully acknowledged for providing the 329 nt RNA.
This work was funded by the German funding agency (DFG) in Collaborative Research Center 902: Molecular principles of RNA-based regulation and in Graduate College: CLIC (GRK1982). Financial support by European access program (iNEXT) is gratefully acknowledged. Harald Schwalbe is member of the DFG-funded Cluster of Excellence: macromolecular complexes (EXC115). Robbin Schnieders is supported by the Fonds of the Chemical Industry. Work at BMRZ is supported by the state of Hesse. We acknowledge access to the 950 MHz spectrometer of the Astbury BioStructure Laboratory BioNMR Facility (University of Leeds) which was funded by the University of Leeds.
- Dingley AJ, Grzesiek S (1998) Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J Am Chem Soc 7863:714–718Google Scholar
- Duchardt-Ferner E, Gottstein-Schmidtke SR, Weigand JE, Ohlenschläger O, Wurm J-P, Hammann C, Suess B, Wöhnert J (2016) What a difference an OH makes: conformational dynamics as the basis for the ligand specificity of the neomycin-sensing riboswitch. Angew Chem Int Ed 55:1527–1530CrossRefGoogle Scholar
- Helmling C, Keyhani S, Sochor F, Fürtig B, Hengesbach M, Schwalbe H (2015) Rapid NMR screening of RNA secondary structure and binding. 63:67–76Google Scholar
- Lu K, Heng X, Garyu L, Monti S, Garcia EL, Kharytonchyk S, Dorjsuren B, Kulandaivel G, Jones S, Hiremath A, Divakaruni SS, Lacotti C, Barton S, Tummillo D, Hosic A, Edme K, Albrecht S, Telesnitsky A, Summers MF (2011) NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging. Science 334:242–245CrossRefADSGoogle Scholar
- Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci 94:12366–12371CrossRefADSGoogle Scholar