TOXIC RNA INHIBITORS SELF-ASSEMBLED IN SITU

Potentmodulators of RNA function can be assembled in cellulo by using the cell as a reaction vessel and a disease-causing RNA as a catalyst. When designing small molecule effectors of function, a balance between permeability and potency must be struck. Low molecular weight compounds are more permeable while higher molecular weight compounds are more potent. The advantages of both types of compounds could be synergized if low molecular weight moleculescould be transformed into potent, multivalent ligands via a reaction catalyzed by bindingto a target in cells expressing a genetic defect. We demonstrate that this approach is indeed viable in cellulo. Small molecule modules with precisely positioned alkyne and azide moieties bind adjacent internal loops in r(CCUG)exp, the causative agent of myotonic dystrophy type 2 (DM2), and are transformed into oligomeric, potent inhibitors of DM2 RNA dysfunction via a 1,3 Huisgen dipolar cycloaddition reaction, a variant of click chemistry. Additionally, we show that this approach is applicable to the r(CUG) repeating RNA that causes myotonic dystrophy type 1 (DM1). The click chemistry approach also allows for FRET sensors to be synthesized on-site by using r(CUG) repeats as a catalyst. Furthermore it is shown that small molecule binding sites in patient-derived cells can be identified by using reactive approaches termed Chem-CLIP and Chem-CLIP-Map. Lastly, it is shown that small molecules that target r(CUG) expansions can be designed to cleave this RNA by appending a smallmolecule with a nucleic acid cleaving module.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. In cellulo, in situclick chemistry to synthesize potent inhibitors of the RNA that causes DM2. A, DM2 is caused by a r(CCUG) repeat that binds and sequesters muscleblind-like 1 protein (MBNL1). Small molecules that contain azide and alkyne functional groups (N.sub.3-K, K-Ak, N.sub.3-K-Ak, and N.sub.3-K-AaK) bind adjacent sites in r(CCUG).sup.exp and undergo a Huisgen dipolar cycloaddition reaction. B, Molecular dynamics (MD) simulation models of clickable modules binding to a mimic of r(CCUG).sup.exp. BI, Conformational searching reveals close proximity between azide and alkyne groups presented by K modules bound to adjacent sites. BII, A low energy state in MD simulation of 1,4-triazole adduct from N.sub.3-K and K-Aak is shown in stereoview. Hydrogen bonds between the RNA and K are shown in dashed lines. BIII, Low energy snapshot of MD simulations showing other linker models.

FIG. 2. Identifying the extent of in cellulo click reactions and the targets of clickable small molecules. A, Schematic of ChemReactBlP, an approach to identify the cellular targets of small molecules. Studies were enabled by using a biotinylated monomer with a single N.sub.3 group to participate in the click reaction, or N.sub.3-K-Biotin, which allows isolation of the clicked oligomer and bound RNA targets by passing cell lysates over streptavidin resin. B, Results of ChemReactBlP mass spectral analysis of pulled down, templated small molecule produ