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.
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.
Max Planck Florida Institute for Neuroscience
