Photocatalytic proximity labeling for the identification of G-quadruplex DNA-interacting proteins

DNA–protein interactions play an essential role in fundamental biological processes in the cell. Herein, we report a photocatalytic proximity labeling approach for the comprehensive identification of G-quadruplex (G4)-interacting proteins (G4IPs) using a photocatalyst-modified human telomere DNA G4 sequence. Ruthenium complex or BODIPY was used as a photocatalyst, and 1-methyl-4-arylurazole (MAUra) was used as a labeling reagent. The initial investigation of the labeling reaction of photocatalyst-modified G4 was conducted using unwinding protein 1 (UP1) as a model of the G4-interacting protein. The SDS-PAGE analysis confirmed the efficient labeling of UP1 with MAUra-azide under photoirradiation. The selective labeling of UP1 was verified using a protein mixture of UP1 and bovine serum albumin. Mass spectrometry revealed that histidine in UP1 was selectively labeled. We utilized this labeling approach for nuclear extract proteins and identified the labeled proteins using quantitative proteomics analysis. Abundant unknown G4 binding protein candidates were identified among these proteins. Notably, hexokinase-1 (HK1) protein, which catalyzes the first, rate-limiting step of glucose metabolism, was identified as a G4IP, and its selective binding toward G4 DNA was confirmed via electrophoretic mobility shift assay. This finding regarding G4 DNA–HK1 protein interaction could be used to highlight the essential subcellular G4-mediated functions of HK1. The proposed labeling approach is a promising tool for investigating the protein interactions of the higher-order structural motifs and functional nucleic acids.