Herein, we describe the design of high affinity ligands that bind expanded rCUG-and rCAG-repeat RNAs expressed in myotonic dystrophy and spinocerebellar ataxia. These ligands also inhibit, with nanomolar IC 50 's, 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 bis-benzimidazole Hoechst 33258, which was identified by searching known RNA-ligand interactions. 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. As 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 multivalent 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 's, the most potent inhibitor is the pentamer, which also inhibits the formation of rCAG repeat-MBNL1 complexes.Comparison of the binding data of 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.

Most ligands targeting RNA are identified through screening a therapeutic target for binding members of a ligand library. A potential alternative way to construct RNA binders is through rational design using information about the RNA motifs ligands prefer to bind. Herein, we describe such an approach to design modularly assembled ligands targeting the RNA that causes myotonic dystrophy type 2 (DM2), a currently untreatable disease. A previous study identified that 6′-N-5-hexynoate kanamycin A (1) prefers to bind 2×2 nucleotide, pyrimidine-rich RNA internal loops. Multiple copies of such loops were found in the RNA hairpin that causes DM2. The 1 ligand was then modularly displayed on a peptoid scaffold with varied number and spacing to target several internal loops simultaneously. Modularly assembled ligands were tested for binding to a series of RNAs and for inhibiting the formation of the toxic DM2 RNA-muscleblind protein (MBNL-1) interaction. The most potent ligand displays three 1 modules, each separated by four spacing submonomers, and inhibits the formation of the RNA-protein complex with an IC 50 of 25 nM. This ligand is higher affinity and more specific for binding DM2 RNA than MBNL-1. It binds the DM2 RNA at least 20-times more tightly than related RNAs and 15-fold more tightly than MBNL-1. A related control peptoid displaying 6′-N-5-hexynoate neamine (2) is >100-fold less potent at inhibiting the RNAprotein interaction and binds to DM2 RNA >125-fold more weakly. Uptake studies into a mouse myoblast cell line also show that the most potent ligand is cell permeable.

Myotonic muscular dystrophy types 1 and 2 (DM1 and DM2, respectively) are caused by expansions of repeating nucleotides in non-coding regions of RNA. In DM1, the expansion is an rCUG triplet repeat whereas the DM2 expansion is an rCCUG quadruplet repeat, both of which fold into hairpin structures with periodically repeating internal loops separated by two 5′GC/3′CG base pairs. The sizes of the loops, however, are different: the DM1 repeat forms 1 × 1 nucleotide UU loops while the DM2 repeat forms 2 × 2 nucleotide 5′CU/3′UC loops. DM is caused when the expanded repeats bind the RNA splicing regulator Muscleblind-like 1 protein (MBNL1), thus compromising its function. Therefore, one potential therapeutic strategy for these diseases is to prevent MBNL1 from binding the toxic RNA repeats. Previously, we designed nanomolar inhibitors of the DM2-MBNL1 interaction by modularly assembling 6′-N-5-hexyonate kanamycin A (K) onto a peptoid backbone. The K ligand binds the 2 × 2 pyrimidine-rich internal loops found in the DM2 RNA with high affinity. The best compound identified from that study contains three K modules separated by four propylamine spacing modules and is 20-fold selective over the DM1 RNA. Because the modularly assembled K-containing compounds also bound the DM1 RNA, albeit with lower affinity, and because the loop size is different, we hypothesized that the optimal DM1 RNA binder may display K modules separated by shorter distance between ligand modules. Indeed, the ideal DM1 RNA binder has only two propylamine spacing modules separating the K ligands. Peptoids displaying three and four K modules on a peptoid scaffold bind the DM1 RNA with Kd's of 20 (3-fold selective for DM1 over DM2) and 4 nM (6-fold selective for DM1 over DM2) and inhibit the RNA-protein interaction with IC50's of 40 and 7 nM, respectively. Importantly, by coupling the two studies together, we have determined that appropriate spacing can affect binding selectivity by 60-fold (20- × 3-fold). The trimer and tetramer also bind ∼13- and ∼63-fold more tightly to DM1 RNAs than does MBNL1. The modularly assembled compounds are cell permeable and non-toxic as determined by flow cytometry. The results establish that for these two systems: (i) a programmable modular assembly approach can provide synthetic ligands for RNA with affinities and specificities that exceed those of natural proteins; and (ii) the spacing of ligand modules can be used to tune specificity for one RNA target over another.