JournalNeural Excitability, Synapses, and Glia

Precise small-molecule recognition of a toxic CUG RNA repeat expansion

DM1 is caused by a toxic gain of function by r(CUG)exp. We designed small molecules that ameliorate disease-associated phenotypes and that can be used to assess target selectivity in DM1 cells. (a) Structure of 1, a non-covalent binding compound. Multivalent small molecules are represented by purple spheres (RNA-binding modules) connected by a line (N-methyl peptide scaffold). Also shown is the secondary structure of r(CUG)exp and binding of MBNL1, which causes disease. Release of MBNL1 by small molecules improved DM1-associated defects. (b) 1 rescued MBNL1 exon 5 splicing defects in DM1-affected cells (n = 6, 6 biological replicates, 2 replicate experiments). (c) 1 reduced the number of r(CUG)exp nuclear foci in DM1 cells (n = 100 cells, 5 biological replicates, 2 replicate experiments). (d) Representative images from RNA-FISH experiments to assess formation of nuclear foci. Scale bars represent 5 μM. Data represent mean values ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, as determined by a two-tailed Student t test.

Excluding the ribosome and riboswitches, developing small molecules that selectively target RNA is a longstanding problem in chemical biology. A typical cellular RNA is difficult to target because it has little tertiary, but abundant secondary structure. We designed allele-selective compounds that target such an RNA, the toxic noncoding repeat expansion (r(CUG)exp) that causes myotonic dystrophy type 1 (DM1). We developed several strategies to generate allele-selective small molecules, including non-covalent binding, covalent binding, cleavage and on-site probe synthesis. Covalent binding and cleavage enabled target profiling in cells derived from individuals with DM1, showing precise recognition of r(CUG)exp. In the on-site probe synthesis approach, small molecules bound adjacent sites in r(CUG)exp and reacted to afford picomolar inhibitors via a proximity-based click reaction only in DM1-affected cells. We expanded this approach to image r(CUG)exp in its natural context.

Rzuczek, S.G., Colgan, L.A., Nakai, Y., Cameron, M.D., Furling, D., Yasuda, R., and Disney, M.D. (2017). Precise small-molecule recognition of a toxic CUG RNA repeat expansion. Nat. Chem. Biol. 13, 188–193.