Transcriptome-Wide Mapping of Small-Molecule RNA-Binding Sites in Cells Informs an Isoform-Specific Degrader of QSOX1 mRNA

Abstract

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound’s molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5′ untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

Pervasive Transcriptome Interactions of Protein-Targeted Drugs

“…Little is known about the RNA off-target interactions of protein-targeted drugs in vivo, especially for clinically approved drugs. Investigation of small molecule-RNA interactions has been carried out recently via photocrosslinking (diazirine) 7,8 , alkylation (chlorambucil) 9,10 , in-line probing 11 , and SHAPE 12 , and these have emerged as useful tools to identify RNA-ligand interactions in vitro and recently in cultured cells 13 . Although elegant, application of these methods in vivo is potentially limited by transcriptional effects during prolonged probe treatment 14 , cellular damage by ultraviolet irradiation 15,16 , and nucleobase biases [17][18][19] .…”

Low-Molecular Weight Small Molecules Can Potently Bind RNA and Affect Oncogenic Pathways in Cells

“…Summary of fragment-based ligand identification strategies for RNA. Previously reported methods for identification of fragment binders for RNA, including NMR spectroscopy, mass spectrometry (MS), equilibrium dialysis, Chem-CLIP-Frag-Map, , and SHAPE probing, compared to this work. Representative examples of fragment hits identified from each method and their affinities for the RNA target are provided.…”

“…However, here, we have shown that compounds of low molecular weight and, hence fragment-like characteristics, should be considered when identifying bioactive chemical matter that binds RNA, affording mechanistically defined small molecules that selectively affect RNA-mediated pathways. It will be interesting to study whether fragments outside the heterocycle space in particular or the currently known RNA-binding space in general have binding capacity and if these interactions can be detected by the methods established herein or others. − Interestingly, some of the fragment hits observed from chemical cross-linking and isolation by pull-down fragment mapping (Chem-CLIP-Frag-Map) suggest that non-heterocyclic compounds also bind RNAs. , …”

“…Previous strategies adopted to study fragment binding of RNA include NMR spectroscopy, mass spectrometry (MS), equilibrium dialysis, Chem-CLIP-Frag-Map, , and selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE) probing (Figure ). For each approach, the initial fragment hit had low affinity for the RNA targethigh μM to mMwhich required further optimization to obtain higher-affinity, selective binders (Table S3).…”

“…For each approach, the initial fragment hit had low affinity for the RNA targethigh μM to mMwhich required further optimization to obtain higher-affinity, selective binders (Table S3). Additionally, these strategies were implemented for a specific RNA target, − , with the exception of transcriptome-wide Chem-CLIP-Frag-Map, although such target-agnostic studies require modification of the fragment library with photo-cross-linking elements. , A recent report from our laboratory showed that DNA-encoded library (DEL) screening could rapidly screen diverse chemical matter for RNA-binding capacity, with subsequent molecular fingerprints generated by 2DCS . Akin to the study herein, these fingerprints were mined to identify targetable structures within the human miRnome, affording an inhibitor of pri-miR-27a that had two binding sites for the small molecule …”