Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

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. DNA-protein interactions are essential to genome function, but they are challenging to map in a cellular environment. Now, a chemical proteomics approach, which uses DNA G-quadruplex-specific ligands containing a photocrosslinking motif, has enabled the systematic identification of DNA G-quadruplex-binding proteins in live cells.

Automated summary

The CMPP strategy allows for the investigation of DNA G-quadruplex (G4) interactions with proteins in native chromatin. This approach enables the identification of G4-interacting proteins in live cells, providing a chemical strategy to study molecular interactions in cellular chromatin.