Detecting the formation of human c-KIT oncogene promoter G-Quadruplex by Taylor dispersion analysis

The formation of G-quadruplex (G4) structures in oncogenic G-rich promoter regions are implicated in their biological functions, especially the inhibition of transcription. The binding of cations is thought to contribute to the stabilization of the G4 formation and competition against the duplex formation in the genomic sequence. Furthermore, it might affect the recognition of DNA-binding proteins. Therefore, measuring the interaction between G4 DNA and cations in a free solution environment is critical for evaluating G4 DNA biological functions. However, how binding to cations (K + and NH 4 + ) affects the folding equilibrium of the G4 structure remains unclear. In this work, a Taylor dispersion analysis (TDA) method using a capillary electrophoresis (CE) instrument was established for the quantitative characterization of the cation-dependent G4 formation in the human c-KIT oncogene promoter region, as well as diffusivities and hydrodynamic radii of DNA variations before and after folding. Our results showed that both K + and NH 4 + can induce the random-coiled c-KIT DNA to unfold and form a more unstretched intermediate state and then fold into tightly structured G4s with smaller size. The G4 size induced by NH 4 + was smaller than that induced by K + ions, though these two cations induced the c-KIT G4 DNA formation with similar binding constants (order of magnitude around 10 6  M −1 ). The TDA method can be widely used for rapid structural analyses of trace amounts of DNA mixtures, which effectively differentiate DNA variations or DNA-ligand complex conformations.