NMR structures and magnetic force spectroscopy studies of small molecules binding to models of an RNA CAG repeat expansion.

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntington's disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG), causes both HD and SCA3, and the RNA's toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, , was discovered that binds to r(CAG) and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5'CAG/3'GAC where the underlined adenosines form a 1×1 nucleotide internal loop) in complex with and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound was designed based on the structure of bound to the RNA while was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loop's closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, and are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes.