DNP-Enhanced Magic Angle Spinning Solid-State NMR Spectroscopy to Determine RNA–Ligand Interactions

Understanding the molecular recognition underlying RNA-ligand complex formation is of key importance to explain the RNA regulatory function of riboswitches and to support the development of low-molecular-weight RNA binders as starting points for the development of RNA-targeting drugs. Here, we report magic angle spinning solid-state NMR spectroscopic studies enhanced by dynamic nuclear polarization (MAS-DNP) to determine the molecular recognition of a ligand-RNA riboswitch complex. We benchmarked different labeling strategies for four large RNAs (70-86 nt) of the aptamer domain of a 2'deoxyguanosine-sensing riboswitch from <i>Mesoplasma florum</i>. RNA samples were prepared either by chemoenzymatic approaches or by solid-phase chemical synthesis employing different labeling schemes of riboswitches of up to 86 nucleotides. RNA-ligand complexes were prepared by the addition of their cognate metabolite. We showed that nucleotide- and ligand-selective labeling are prerequisites for the MAS-DNP studies to reduce the NMR signal overlap present in such large RNAs. We further extended site-specific labeling to atom-specific labeling, which allowed us to derive the structure of the ligand-binding pocket, extending the application of 2D ¹³C,¹⁵N-TEDOR experiments. The work described here opens an avenue for the investigation of large RNA-ligand complexes by MAS-DNP.