Degradation of G‐Quadruplex‐Binding Proteins by G4L‐PROTAC via Quaternary Complex Formation

Abstract

Abstract G‐Quadruplexes (G4s) are noncanonical nucleic acid secondary structures enriched in genomic regions critical for transcription and replication. These dynamic scaffolds recruit G4‐binding proteins (G4BPs), thereby regulating diverse cellular processes. However, the functional roles of G4BPs in the G4‐bound state remain poorly defined. Here, we report the development of G4L‐PROTACs—bifunctional small molecules that couple a G4 ligand with an E3 ligase recruiter to achieve selective proteasomal degradation of G4‐bound G4BPs. Unlike RNAi or CRISPR‐Cas9, which eliminate proteins irrespective of binding state, G4L‐PROTACs enable depletion of G4BPs only when associated with G4s. Using model G4 motifs from telomeres and the NRAS 5′ UTR, we demonstrated in vitro ternary complex formation. In cells, G4L‐PROTAC treatment reduced endogenous levels of the G4‐resolving helicase DHX36, resulting in a marked increase in intracellular G4 abundance, as shown by BG4 immunofluorescence. This phenotype highlights the ability of G4L‐PROTACs to modulate the G4–protein equilibrium in living cells. Notably, G4L‐PROTACs do not induce G4‐mediated transcriptional silencing, underscoring their precision in modulating nucleic acid–protein interactions. This strategy offers a powerful platform for probing G4–G4BP functions and holds promise for therapeutic targeting of G4‐associated proteins.