A simple ligand that selectively targets CUG trinucleotide repeats and inhibits MBNL protein binding

This work describes the rational design, synthesis, and study of a ligand that selectively complexes CUG repeats in RNA (and CTG repeats in DNA) with high nanomolar affinity. This sequence is considered a causative agent of myotonic dystrophy type 1 (DM1) because of its ability to sequester muscleblind-like (MBNL) proteins. Ligand 1 was synthesized in two steps from commercially available compounds, and its binding to CTG and CUG repeats in oligonucleotides studied. Isothermal titration calorimetry studies of 1 with various sequences showed a preference toward the T-T mismatch (Kd of 390 +/- 80 nM) with a 13-, 169-, and 85-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Binding and Job analysis of 1 to multiple CTG step sequences revealed high affinity binding to every other T-T mismatch with negative cooperativity for proximal T-T mismatches. The affinity of 1 for a (CUG) 4 step provided a Kd of 430 nM with a binding stoichiometry of 1:1. The preference for the U-U in RNA was maintained with a 6-, >143-, and >143-fold reduction in affinity toward single C-C, A-A, and G-G mismatches, respectively. Ligand 1 destabilized the complexes formed between MBNL1N and (CUG)(4) and (CUG)(12) with IC50 values of 52 +/- 20 mu M and 46 +/- 7 mu M, respectively, and K-i values of 6 +/- 2 mu M and 7 +/- 1 mu M, respectively. These values were only minimally altered by the addition of competitor tRNA. Ligand 1 does not destabilize the unrelated RNA-protein complexes the U1A-SL2 RNA complex and the Sex lethal-tra RNA complex. Thus, ligand 1 selectively destabilizes the MBNL1N-poly(CUG) complex.