Biomolecular NMR Spectroscopy of Ribonucleic Acids

Abstract

Biomolecular nuclear magnetic resonance (NMR) spectroscopy allows the characterisation of structural and dynamic properties of ribonucleic acids (RNAs) in solution. The NMR‐based determination of high‐resolution three‐dimensional (3D) structures by NMR spectroscopy in solution is especially useful for small‐to‐medium sized RNA molecules like aptamers and small ribozymes, but has also been achieved for RNAs up to about 100 nucleotides in total size. Biomolecular NMR also provides valuable information about the interaction between RNA and diverse binding partners such as drugs, peptides, proteins or other nucleic acids. In addition, novel methods can be utilised to characterise the role of metal ions, intramolecular dynamics across a range of motion time scales and shifted pKa values of exchangeable nucleobase protons in RNA structure and catalysis.

Key Concepts:

  • High‐resolution NMR spectroscopy in solution is a powerful method to determine the 3D structures of small and medium sized RNA molecule.

  • The structure determination of larger RNAs generally requires labelling with stable isotopes

  • The information about molecular dynamics that can be obtained by NMR experiments allows quantitative studies of molecular motion across a wide range of time scales.

  • The possibility to directly observe changes in the pKa values of ionisable groups.

  • The localisation of divalent metal ions can be defined using several NMR methods.

Keywords: RNA structure; isotope labelling; molecular dynamics; metal–ion binding; RNA synthesis; Dynamic NMR spectroscopy; Chemical Shift

Figure 1.

Diagram outlining a general approach for preparation of an nuclear magnetic resonance (NMR) sample of ribonucleic acid (RNA).

Figure 2.

Diagram describing the stepwise procedure for solution structure determination of ribonucleic acid (RNA) by nuclear magnetic resonance (NMR) spectroscopy.

Figure 3.

Resonance assignment of imino protons based on the secondary structure of the RNA. (a) G and U imino protons stabilized by the formation of Watson–Crick U–A and G–C base pairs give strong NOE signals to specific atoms of the paired residue (pink lines) that allow their identification. (b) Secondary structure of the SLV (stem‐loop V) RNA derived from the Neurospora VS (Varkud satellite) ribozyme (Campbell and Legault, ) outlining the NOE connectivities between imino protons of adjacent base pairs in the secondary structure (pink lines). (c) Imino region of the 2D NOESY spectrum of SLV showing the path of NOE connectivities (pink lines) between imino protons of adjacent base pairs. The imino protons of the two base pairs at each end of the stem (G1‐C17 and U6‐A12) are not observed in this spectrum due to the dynamics of these base pairs.

Figure 4.

Sequential assignment of nonexchangeable protons in RNA. (a) Secondary structure of the SLI′ (stem‐loop I) RNA derived from the Neurospora VS ribozyme (Hoffmann et al., ). (b) Dinucleotide fragment (C4‐G5) extracted from the 3D structure of SLI′ (PDB entry: 1OW9) showing the short intra‐ and internucleotide H6/H8‐H1′ distances (pink dotted lines). (c) Region of the 2D NOESY spectrum of SLI′ showing the path of NOE connectivities (pink lines) for the sequential walk between residues 4 and 11 (see text). The intranucleotide H6/H8‐H1′ NOE signals are annotated by the residue numbers.

Figure 5.

(a) Ribose structure and ribose proton nomenclature. (b) Ribose region of the 2D 1H–13C correlation spectrum of stem‐loop I (SLI′) showing the increased signal resolution provided by the 13C chemical shift.

Figure 6.

Identification of metal‐binding sites in RNA by NMR methods (Bonneau and Legault, ). (a) 1D 31P spectrum of the stem‐loop VI (SLVI) RNA derived from the Neurospora VS ribozyme and containing a single phosphorothioate modification at the nonbonded pro‐Rp phosphate oxygen of residue A15 (A15‐Rp). The pink arrow points to the 31P signal of the modified phosphate. The inset shows the effect of cadmium on the downfield region of the 1D 31P spectrum of A15‐Rp as well as the A15‐Sp isomer. The RNA samples contained either 5 mM MgCl2:0 mM CdCl2 or 4.75 mM MgCl2:0.25 mM CdCl2. (b) Binding of a divalent metal ion (grey sphere) to the GAAA tetraloop from the NMR structure of SLVI (PDB entry: 2MIS) determined using restraints derived from cadmium titration of phosphorothioate RNA and manganese‐induced PRE. The modified oxygens in A15‐Rp (pink) and A15‐Sp (blue) are highlighted as well as inner‐sphere metal binding (dotted blue line) and atoms that are most affected by manganese‐induced PRE (green).

Figure 7.

Time scales of dynamic processes found in RNA and the NMR methods that can be used to study them.

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Dieckmann, Thorsten, Piazza, Michael, Bonneau, Eric, and Legault, Pascale(Jul 2014) Biomolecular NMR Spectroscopy of Ribonucleic Acids. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021033.pub2]