Photoactive G‑Quadruplex Ligand Identifies Multiple G‑Quadruplex-Related Proteins with Extensive Sequence Tolerance in the Cellular Environment

G-Quadruplex (G4) is a noncanonical
nucleic acid secondary structure
with multiple biofunctions. Identifying G4-related proteins (G4RPs)
is important for understanding the roles of G4 in biology. Current
methods to identify G4RPs include discovery from specific biological
processes or in vitro pull-down assays with specific
G4 sequences. Here, we report an in vivo strategy
used to identify G4RPs with extensive sequence tolerance based on
G4 ligand-mediated cross-linking. Applying this method, we identified
114 and 281 G4RPs in SV589 and MM231 cells, respectively. The results
successfully overlapped with all the pull-down assay literature. Through
the electrophoretic mobility shift assay (EMSA), we identified some
new G4-binding proteins. Moreover, enhanced cross-linking and immunoprecipitation
(eCLIP) confirmed that one newly identified G4-binding protein, SERBP1,
interacts with G4 in the cellular environment. The method we developed
provides a new strategy for identifying proteins that interact with
nucleic secondary structures in cells and benefit the study of their
biological roles.