RNA G-quadruplex structure-based PROTACs for targeted DHX36 protein degradation and gene activity modulation in mammalian cells

RNA G-quadruplexes (rG4s) are non-canonical secondary nucleic acid structures found in the transcriptome. They play crucial roles in gene regulation by interacting with G4-binding proteins (G4BPs) in cells. rG4-G4BP complexes have been associated with human diseases, making them important targets for drug development. Generating innovative tools to disrupt rG4-G4BP interactions will provide a unique opportunity to explore new biological mechanisms and potentially treat related diseases. Here, we have rationally designed and developed a series of rG4-based proteolytic targeting chimeras (rG4-PROTACs) aimed at degrading G4BPs, such as DHX36, a specific G4BP that regulates gene expression by binding to and unraveling rG4 structures in messenger RNAs (mRNAs). Our comprehensive data and systematic analysis reveals that rG4-PROTACs predominantly and selectively degrade DHX36 through a proteosome-dependent mechanism, which promotes the formation of the rG4 structure in mRNA, leading to the translation inhibition of rG4-containing transcripts. Notably, rG4-PROTACs inhibit rG4-mediated APP protein expression, and impact the proliferative capacity of skeletal muscle stem cells by negatively regulating Gnai2 protein expression. In summary, rG4-PROTACs provide a new avenue to understand rG4-G4BP interactions and the biological implications of dysregulated G4BPs, promoting the development of PROTACs technology based on the non-canonical structure of nucleic acids. © The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.