Photoactivatable, Biomimetic Ligand photoMultiTASQ Traps DNA/RNA G‑Quadruplexes and Their Protein Binding Partners

The biological role of DNA and RNA G-quadruplexes (G4s) is relayed to the cellular circuitry by a plethora of proteins known as G4-binding proteins (G4PBs). It is thus critical to decipher the formation of the G4/G4BP complexes to accurately understand their involvement in G4-mediated regulatory mechanisms. While hundreds of G4-interacting compounds (G4 ligands) have been used to uncover G4 biology in a rather indirect manner, only a handful of multivalent ligands allowing for identifying associated proteins have been designed, mostly relying on the covalent capture of G4BPs upon photoactivation. We report herein on such a multivalent ligand named photoMultiTASQ, which belongs to a family of biomimetic and smart G4 ligands known as template-assembled synthetic G-quartets (TASQs). We show how photoMultiTASQ interacts with and photo-cross-links both DNA/RNA G4s and related G4BPs, and develop a new mass spectrometry (MS)-based technique to characterize the covalent adducts. Collectively, these results make photoMultiTASQ a new molecular tool in the arsenal of chemical cross-linking and isolation by pull-down (Chem-CLIP) technologies that uniquely identify targets and validate target engagement in cells.