Herein, we describe the design of high affinity
ligands that bind expanded rCUG and rCAG repeat RNAs expressed in
myotonic dystrophy type 1 (DM1) and
spinocerebellar ataxia type 3. These
ligands also inhibit, with nanomolar IC(50) values, the formation of
RNA-
protein complexes that are implicated in both disorders. The expanded rCUG and rCAG repeats form stable
RNA hairpins with regularly repeating internal loops in the stem and have deleterious effects on cell function. The
ligands that bind the repeats display a derivative of the
bisbenzimidazole Hoechst 33258, which was identified by searching known
RNA-
ligand interactions for
ligands that bind the internal loop displayed in these hairpins. A series of 13 modularly assembled
ligands with defined valencies and distances between
ligand modules was synthesized to target multiple motifs in these RNAs simultaneously. The most avid binder, a pentamer, binds the rCUG repeat hairpin with a K(d) of 13 nM. When compared to a series of related RNAs, the pentamer binds to rCUG repeats with 4.4- to >200-fold specificity. Furthermore, the affinity of binding to rCUG repeats shows incremental gains with increasing valency, while the background binding to genomic
DNA is correspondingly reduced. Then, it was determined whether the modularly assembled
ligands inhibit the recognition of
RNA repeats by Muscleblind-like 1 (MBNL1)
protein, the expanded-rCUG
binding protein whose sequestration leads to splicing defects in DM1. Among several compounds with nanomolar IC(50) values, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes. Comparison of the binding data for the designed synthetic
ligands and MBNL1 to repeating RNAs shows that the synthetic
ligand is 23-fold higher affinity and more specific to DM1 RNAs than MBNL1. Further studies show that the designed
ligands are cell permeable to mouse myoblasts. Thus, cell permeable
ligands that bind repetitive RNAs have been designed that exhibit higher affinity and specificity for binding
RNA than natural
proteins. These studies suggest a general approach to targeting
RNA, including those that cause
RNA dominant disease.