High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA

We present a high-resolution nuclear magnetic resonance (NMR) solution structure of a 14-mer RNA hairpin capped by cUUCGg tetraloop. This short and very stable RNA presents an important model system for the study of RNA structure and dynamics using NMR spectroscopy, molecular dynamics (MD) simulations and RNA force-field development. The extraordinary high precision of the structure (root mean square deviation of 0.3 Å) could be achieved by measuring and incorporating all currently accessible NMR parameters, including distances derived from nuclear Overhauser effect (NOE) intensities, torsion-angle dependent homonuclear and heteronuclear scalar coupling constants, projection-angle-dependent cross-correlated relaxation rates and residual dipolar couplings. The structure calculations were performed with the program CNS using the ARIA setup and protocols. The structure quality was further improved by a final refinement in explicit water using OPLS force field parameters for non-bonded interactions and charges. In addition, the 2′-hydroxyl groups have been assigned and their conformation has been analyzed based on NOE contacts. The structure currently defines a benchmark for the precision and accuracy amenable to RNA structure determination by NMR spectroscopy. Here, we discuss the impact of various NMR restraints on structure quality and discuss in detail the dynamics of this system as previously determined.     

RNA phosphodiester backbone dynamics of a perdeuterated cUUCGg tetraloop RNA from phosphorus-31 NMR relaxation analysis

We have analyzed the relaxation properties of all ³¹P nuclei in an RNA cUUCGg tetraloop model hairpin at proton magnetic field strengths of 300, 600 and 900 MHz in solution. Significant H, P dipolar contributions to R ₁ and R ₂ relaxation are observed in a protonated RNA sample at 600 MHz. These contributions can be suppressed using a perdeuterated RNA sample. In order to interpret the ³¹P relaxation data (R ₁, R ₂), we measured the ³¹P chemical shift anisotropy (CSA) by solid-state NMR spectroscopy under various salt and hydration conditions. A value of 178.5 ppm for the ³¹P CSA in the static state (S ² = 1) could be determined. In order to obtain information about fast time scale dynamics we performed a modelfree analysis on the basis of our relaxation data. The results show that subnanosecond dynamics detected around the phosphodiester backbone are more pronounced than the dynamics detected for the ribofuranosyl and nucleobase moieties of the individual nucleotides (Duchardt and Schwalbe, J Biomol NMR 32:295–308, 2005; Ferner et al., Nucleic Acids Res 36:1928–1940, 2008). Furthermore, the dynamics of the individual phosphate groups seem to be correlated to the 5′ neighbouring nucleobases.