Linker Optimization of cMYC RIBOTAC for Efficient cMYC Modulation

Ribonuclease‐targeting chimeras (RIBOTACs) are heterobifunctional molecules composed of an RNA‐binding motif and an RNase L–recruiting module, enabling selective RNA cleavage by recruited RNase L. While optimization efforts have primarily focused on RNA‐binding ligands and RNase L‐recruiting motifs, the linker design remains poorly understood. Here, we systematically evaluated the impact of linker length and rigidity in a cMYC‐targeting RIBOTAC system. A series of PEG‐based linkers (PEG2–PEG7) was employed while maintaining identical RNA‐binding and RNase L–recruiting modules. Functional evaluation through in vitro cleavage, RT‐qPCR, and Western blot analysis established PEG3 and PEG4 as the optimal linkers, whereas further elongation to PEG5‐7 or substitution with a rigid triazole‐based linker resulted in complete loss of activity. These findings demonstrate that precise linker design is essential for effective RNA degradation.