Transcriptome-Wide, Unbiased Profiling of Ribonuclease Targeting Chimeras

Area of Science:

• Molecular Biology
• RNA Therapeutics
• Drug Discovery

Background:

• Targeting RNA offers a therapeutic strategy to modulate gene expression.
• Small molecules can bind RNA, but inducing targeted cleavage remains challenging.
• Heterobifunctional molecules, like RiboTACs, recruit enzymes to cleave RNA targets.

Purpose of the Study:

• To investigate the correlation between small molecule binding and RNA cleavage induced by RiboTACs.
• To identify factors governing the efficiency of RiboTAC-mediated RNA cleavage.
• To provide insights for designing effective RiboTAC-based therapeutics.

Main Methods:

• Transcriptome-wide analysis of RiboTAC-induced RNA cleavage.
• Global analysis of RNA structure, RNase L cleavage sites, and target expression levels.
• Case study using a RiboTAC targeting LGALS1 (galectin-1) mRNA in cancer cells.
• Assessment of linker length, cellular uptake, and RNase L recruitment module impact.

Main Results:

• Only a subset of RiboTAC-bound RNA targets were cleaved by RNase L.
• Cleaved targets often feature stable structures around the binding site and nearby RNase L cleavage sites.
• RiboTACs facilitate target-enzyme interaction, with target expression influencing cleavage extent.
• A LGALS1-targeting RiboTAC reduced galectin-1 protein and affected cancer cell phenotypes, unlike the binder alone.

Conclusions:

• RiboTAC design requires consideration of RNA structure, RNase L site proximity, and target expression.
• Optimizing linker length and RNase L recruitment enhances RiboTAC potency.
• These findings facilitate the rational design of RiboTACs for therapeutic applications.