1. Chemical profiling of DNA G-quadruplex-interacting proteins in live cells
- Author
-
Shankar Balasubramanian, Xiaoyun Zhang, Santosh Adhikari, Sergio Martínez Cuesta, Jochen Spiegel, Zhang, Xiaoyun [0000-0002-4132-3573], Spiegel, Jochen [0000-0001-7641-1066], Martínez Cuesta, Sergio [0000-0001-9806-2805], Adhikari, Santosh [0000-0002-1501-2106], Balasubramanian, Shankar [0000-0002-0281-5815], and Apollo - University of Cambridge Repository
- Subjects
Models, Molecular ,Ultraviolet Rays ,General Chemical Engineering ,Data_MISCELLANEOUS ,MathematicsofComputing_GENERAL ,Computational biology ,639/638/92/555 ,010402 general chemistry ,G-quadruplex ,Proteomics ,Ligands ,01 natural sciences ,Interactome ,Proof of Concept Study ,Protein–protein interaction ,03 medical and health sciences ,chemistry.chemical_compound ,Target identification ,Nucleic Acids ,Cell Line, Tumor ,Humans ,ComputingMilieux_MISCELLANEOUS ,030304 developmental biology ,0303 health sciences ,Binding protein ,article ,639/638/92/96 ,General Chemistry ,DNA ,Chromatin ,0104 chemical sciences ,Protein-protein interaction networks ,639/638/92/475/2290 ,DNA-Binding Proteins ,G-Quadruplexes ,Cross-Linking Reagents ,HEK293 Cells ,chemistry ,Diazomethane ,Alkynes ,Nucleic acid ,Aminoquinolines ,Nucleic Acid Conformation ,631/337/100 ,Chemical tools ,Protein Binding - Abstract
Funder: Welcome Trust (209441/Z/17/Z), DNA–protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.
- Published
- 2021
- Full Text
- View/download PDF