This study reports the development and application of an engineered, structure-specific antibody (BG4) to visualize DNA G-quadruplex structures in human cells. G-quadruplexes, formed by four guanines held together by Hoogsteen hydrogen bonding, are highly stable under near-physiological conditions and have been implicated in various cellular processes such as transcription, recombination, and replication. The authors used phage display to isolate BG4, which binds with high affinity and selectivity to G-quadruplex structures. They demonstrated that G-quadruplex formation is modulated during the cell cycle, increasing during the S phase when DNA replication occurs. Additionally, they showed that a small molecule ligand, pyridostatin (PDS), can stabilize G-quadruplex structures in cells, leading to an increase in the number of G-quadruplex foci detected by BG4. These findings provide evidence for the formation of G-quadruplex structures in mammalian cells and highlight the potential of stabilizing ligands to target and intervene with their function.This study reports the development and application of an engineered, structure-specific antibody (BG4) to visualize DNA G-quadruplex structures in human cells. G-quadruplexes, formed by four guanines held together by Hoogsteen hydrogen bonding, are highly stable under near-physiological conditions and have been implicated in various cellular processes such as transcription, recombination, and replication. The authors used phage display to isolate BG4, which binds with high affinity and selectivity to G-quadruplex structures. They demonstrated that G-quadruplex formation is modulated during the cell cycle, increasing during the S phase when DNA replication occurs. Additionally, they showed that a small molecule ligand, pyridostatin (PDS), can stabilize G-quadruplex structures in cells, leading to an increase in the number of G-quadruplex foci detected by BG4. These findings provide evidence for the formation of G-quadruplex structures in mammalian cells and highlight the potential of stabilizing ligands to target and intervene with their function.