Figure 6. Target validation and profiling tools enabled by SMIRNAs. SMIRNAs have been developed that cross-link with their cellular targets (Chem-CLIP), cleave their cellular targets (RiboSNAP), change target RNA sequences, or compete with ASOs for binding (ASO-Bind-Map). The methods are complementary, and the ideal method to employ will depend on the RNA target and the inherent sequence specificity of the cross-linking, cleavage, or reactive species generated. (A) Chem-CLIP, a cross-linking approach in which small molecules bind to RNA targets and undergo a proximity-based reaction at the binding site, tagging the RNA with a purification tag. (B) Ribo-SNAP, a cleavage-based approach in which small molecules bind to RNA targets in cells and undergo a proximity-based cleavage reaction at the binding site, allowing transcriptome-wide assessment of target engagement. (C) Changing RNA sequence with a small molecule. A small molecule that targets an RNA is appended with ruthenium bipyridine. Irradiation of cells and animals with light produces reactive oxygen species that convert G to *-oxo-G. Recognition of 8-oxo-G lesions by antibodies allows immunoprecipitation of bound RNAs, which can then be analyzed. (D) ASO-Bind-Map is a competition-based experiment between ASOs and small molecules. The binding of small molecules thermodynamically stabilizes a region of defined structure and inhibits ASO binding. Inhibition of ASO cleavage indicates SMIRNA binding of the targeted mRNA. (E) On-site drug synthesis can be used to study RNA target engagement. Briefly, two pro-drugs harboring a complementary donor or acceptor bind adjacent structures in an RNA target, triggering a proximity-based click reaction (RNA is the catalyst) and producing a FRET signal. This allows imaging target engagement and also tracking of the target upon binding to the drug.

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