Single-Molecule Detection on Intrastrand Interactions among G4 Clusters

G-quadruplexes (G4s) are noncanonical DNA structures that regulate gene expression by serving as mechanical barriers. Telomeric DNA and oncogenic promoter regions (e.g., c-KIT and h-RAS) often contain multiple G-quadruplex structures, which may regulate the transcription of adjacent genes through their interactions. These G4 clusters hold significant therapeutic potential as drug targets for cancer treatment. Thus, understanding the interactions within G4 clusters is of considerable importance. However, current bulk measurements struggle to elucidate the dynamic interactions among multiple intrastranded G4s due to the presence of potential interstrand interactions. Herein, we establish a single-molecule platform to investigate the intrastrand interactions between G4s in the c-KIT promoter region. Our results reveal that these G4 units interact to form higher-order structures, with kit* acting as a key regulator that couples and influences the formation and stability of adjacent G4 units. This single-molecule platform can also detect interactions between small-molecule ligands and consecutive G4 clusters. Compared to CX-5461, PDS is a more effective inhibitor of c-KIT. This single-molecule detection method provides robust support for investigating the intrastrand interactions between consecutive G4 structures.