Epigenetic mutations: Effects on the stability of G-quadruplex structures in the c-KIT promoter

G-quadruplexes (G4s) are non-canonical DNA structures that form in guanine-rich genomic regions and play key roles in the regulation of gene expression. These structures are frequently located within promoter regions and often overlap CpG islands, making them particularly sensitive to epigenetic modifications that can influence their stability and biological function. The promoter of the oncogene c-KIT contains three G4-forming sequences; among them, c-KIT1 was selected as a model system because it includes cytosines positioned near guanines involved in G-tetrad formation, providing an ideal context to probe how local epigenetic changes influence G4 architecture. The aim of this study was to determine how specific cytosine modifications affect G4 structure, thermodynamic stability, and ligand interactions. To this end, c-KIT1 and six related variants bearing epigenetic modifications, specifically 5-methylcytosine or 5-carboxylcytosine at defined positions, were investigated. Mass spectrometry experiments confirmed that all modified sequences maintain a parallel G4 topology. Circular Dichroism Spectroscopy (CD) and Differential Scanning Calorimetry (DSC) were then employed to evaluate G4 formation and the thermodynamic stability of all investigated G4s. To determine whether epigenetic cytosine modifications influence G4-ligand interactions, CD melting experiments were also performed in the presence of a panel of HDAC inhibitors. Among the tested compounds, one ligand produced the most pronounced increase in G4 thermal stability, identifying it as a promising candidate for selective G4 targeting.