Precise Small Molecule Degradation of a Noncoding RNA Identifies Cellular Binding Sites and Modulates an Oncogenic Phenotype.

Herein, we describe the precise cellular destruction of an oncogenic noncoding RNA with a small molecule-bleomycin A5 conjugate, affording reversal of phenotype and a facile method to map the cellular binding sites of a small molecule. In particular, bleomycin A5 was coupled to a small molecule that selectively binds the microRNA-96 hairpin precursor (pri-miR-96). By coupling of bleomycin A5's free amine to the RNA binder, its affinity for binding to pri-miR-96 is >100-fold stronger than to DNA and the compound selectively cleaves pri-miR-96 in triple negative breast cancer (TNBC) cells. Indeed, selective cleavage of pri-miR-96 enhanced expression of FOXO1 protein, a pro-apoptotic transcription factor that miR-96 silences, and triggered apoptosis in TNBC cells. No effects were observed in healthy breast epithelial cells. Thus, conjugation of a small molecule to bleomycin A5's free amine may provide programmable control over its cellular targets. Few approaches are available to define the binding sites of small molecules within cellular RNAs. Our targeted cleavage method provides such an approach that is straightforward to implement. That is, we determined experimentally the site cleaved within pri-miR-96 in vitro and in cells; these studies revealed that the site of cleavage is the precise site for which the small molecule cleaver was designed and in agreement with modeling. These studies demonstrate the potential of sequence-based design to provide bioactive compounds that precisely recognize and cleave RNA in cells.