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

Myotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy that is caused by an expanded repeat of r(CCUG) [r(CCUG)] in intron 1 of a CHC-type zinc finger nucleic acid binding protein () 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). Herein, we study the biological impacts of the molecular recognition of r(CCUG)'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) sequence for cleavage exacerbate DM2 defects due to recognition of a short r(CCUG) sequence that is embedded in , argonaute-1 (), and , reducing their levels. The latter event causes a greater depletion of functional MBNL1 than the amount already sequestered by r(CCUG). Thus, compounds targeting RNA structures can have functional advantages over oligonucleotides that target the sequence in some disease settings, particularly in DM2.