Structure-Specific Cleavage of an RNA Repeat Expansion with a Dimeric Small Molecule Is Advantageous over...

Myotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy that is caused by an expanded repeat of r(CCUG) [r(CCUG)^exp] in intron 1 of a CHC-type zinc finger nucleic acid binding protein (CNBP) pre-mRNA. Various mechanisms contribute to DM2 pathology including pre-mRNA splicing defects caused by sequestration of the RNA splicing regulator muscleblind-like-1 (MBNL1) by r(CCUG)^exp. Herein, we study the biological impacts of the molecular recognition of r(CCUG)^exp's structure by a designer dimeric small molecule that directly cleaves the RNA in patient-derived cells. The compound is comprised of two RNA-binding modules conjugated to a derivative of the natural product bleomycin. Careful design of the chimera affords RNA-specific cleavage, as attachment of the bleomycin cleaving module was done in a manner that disables DNA cleavage. The chimeric cleaver is more potent than the parent binding compound for alleviating DM2-associated defects. Importantly, oligonucleotides targeting the r(CCUG)^exp sequence for cleavage exacerbate DM2 defects due to recognition of a short r(CCUG) sequence that is embedded in CNBP, argonaute-1 (AGO1), and MBNL1, reducing their levels. The latter event causes a greater depletion of functional MBNL1 than the amount already sequestered by r(CCUG)^exp. Thus, compounds targeting RNA structures can have functional advantages over oligonucleotides that target the sequence in some disease settings, particularly in DM2.