Insights into the development of chemical probes for RNA

Morgan, Brittany S; Forte, Jordan E; Hargrove, Amanda E

In contrast to small molecules, most multivalent ligands were developed through rational design based on the secondary structure of the RNA target [ Figure 5A ]. Generally, development began by the identification of monovalent ligands that bound to a particular secondary structure motif(s) by screening, literature search, or using Inforna [See section 'Other Methods of Small Molecule Discovery']. Monomers were covalently linked by selecting and optimizing appropriate sized spacers. For several multivalent ligands, the design was inspired by a crystal structure of r(CUG) sequences, leading to ligands targeting r(CUG) exp in Drosophila melanogaster models (66) . This approach linked acridine and a triaminotriazine unit, the latter of which was proposed to recognize the non-optimal base pairing of U-U mismatches by Janus-wedge hydrogen bonding. Stacking of the two units was expected to decrease nonspecific intercalative binding. This early design was optimized to yield bisamidinium conjugates that mitigated the glossy and rough eye phenotype observed in a DM1 transgenic Drosophila melanogaster model (67) . A different approach utilized Hoechst 33258, which had been previously reported to bind a 5 CUG/3 GUC internal loop (68) . Hoechst 33258 was modified to contain an azide handle and then covalently linked to a peptoid backbone via click chemistry (21) . After extensive linker optimization, multivalent ligands were identified that improved r(CUG) exprelated splicing defects in a mouse model of DM1.