Single-molecule approaches to study G-quadruplex, R-loop, and protein interactions

G-quadruplexes (G4) are non-canonical nucleic acid structures formed by stacked Hoogsteen base-paired guanine (G) quartets, stabilized by monovalent cations. Due to the inherent propensity of G-rich sequences to fold into G4 structures, both DNA and RNA G4s are widely detected in cells. Previous studies have demonstrated that the unique structure and exceptional stability of G4s can act as physical barriers to translocating enzymes and modulate nucleic acid topology. In addition, G4-binding proteins (G4BPs), including G4 helicases, interact with G4 structures and modulate their stability, thereby contributing to the regulation of transcription and translation. This review highlights single-molecule approaches for probing G4-G4BP interactions and recent advances elucidating G4 functions in the R-loop and transcription.