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

TL;DR

This paper uses advanced NMR techniques to study how RNA interacts with small molecules, which could help in developing RNA-targeting drugs.

Contribution

The study introduces MAS-DNP as a novel method to investigate RNA-ligand interactions in large RNA complexes.

Findings

MAS-DNP enables detailed analysis of RNA-ligand interactions in large RNA complexes.

Selective labeling strategies are essential to reduce NMR signal overlap in large RNAs.

Site-specific and atom-specific labeling allows structural insights into ligand-binding pockets.

Abstract

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 Mesoplasma florum. 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.

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