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Updated: Oct 30, 2025
Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes
Published on: April 4, 2025
Detecting the formation of human c-KIT oncogene promoter G-Quadruplex by Taylor dispersion analysis
Yunhe Yang1, Yang Yang1, Shuangshuang Wang1
1National and Local Joint Engineering Research Center of Biomedical Functional Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Changzhou Institute of Innovation and Development, School of Chemistry and Materials Science, Nanjing Normal University, Nanjing, 210023, PR China.
Abstract:
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 NH4+) 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 NH4+ 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 NH4+ 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 106 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.
Insights
Cations like potassium (K+) and ammonium (NH4+) stabilize G-quadruplex (G4) DNA structures in oncogenes. This study quantizes cation-induced G4 formation and structural changes using Taylor dispersion analysis.
Area of Science:
- Molecular Biology
- Biophysical Chemistry
- Genomics
Background:
- G-quadruplex (G4) structures in oncogenic promoter regions are crucial for biological functions, including transcription inhibition.
- Cation binding, particularly K+ and NH4+, is known to stabilize G4 formation, influencing DNA structure and protein interactions.
- The precise impact of cation binding on the folding equilibrium of G4 structures remains incompletely understood.
Purpose of the Study:
- To establish and apply Taylor dispersion analysis (TDA) for quantitative characterization of cation-dependent G-quadruplex (G4) formation.
- To investigate the effects of K+ and NH4+ on the folding equilibrium, diffusivities, and hydrodynamic radii of G4 structures in the human c-KIT oncogene promoter region.
- To elucidate the structural differences in G4 formation induced by different cations.
Main Methods:
- Development of a Taylor dispersion analysis (TDA) method utilizing capillary electrophoresis (CE).
- Quantitative analysis of cation-dependent G4 formation in the human c-KIT oncogene promoter DNA.
- Measurement of DNA diffusivities and hydrodynamic radii before and after cation-induced folding.
Main Results:
- Both K+ and NH4+ induce the unfolding of random-coiled c-KIT DNA into intermediate states and subsequently into tightly structured G4s.
- G4 structures formed with NH4+ exhibit a smaller hydrodynamic size compared to those formed with K+.
- Similar binding constants (around 10^6 M^-1) were observed for both K+ and NH4+ in inducing c-KIT G4 DNA formation.
Conclusions:
- Taylor dispersion analysis (TDA) is an effective method for rapid structural analysis of DNA and DNA-ligand complexes, even with trace amounts.
- Cations differentially affect the size of G-quadruplex structures, with NH4+ inducing smaller G4s than K+.
- The TDA method provides valuable insights into cation-dependent G4 formation and can differentiate DNA variations and conformational changes.
