Photoclick chemistry led to the identification of HELLS as a helicase for DNA G-quadruplexes.

Guanine quadruplexes (G4s) are unique secondary structures of nucleic acids with functions in many biological processes. Understanding the functions of DNA G4s requires knowledge about their recognition by cellular proteins. Here, we developed a method involving photoclick chemistry and LC-MS/MS-based quantitative proteomics for uncovering G4-binding proteins (G4BPs). By incorporating a photoactivatable ortho-nitrobenzylamine moiety into G4 DNA probes and employing UVA irradiation along with stringent washing, we identified 99 proteins enriched with G4 structures derived from the human telomere. By employing fluorescence anisotropy, fluorescence resonance energy transfer, immunofluorescence microscopy, and ChIP-seq analyses, we demonstrated the abilities of one of these proteins, HELLS, in binding and resolving G4 structures in vitro and in chromatin. We also found that HELLS-G4 interaction modulates gene expression. Together, we developed a photoclick chemistry-based approach for identifying G4BPs. The approach allowed for the identification of a number of new G4BPs, and we also documented novel functions of one of these proteins, i.e. HELLS, in recognizing and unwinding G4 structures in vitro and in cells.