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.