Beyond Structure: Methylation Fine‐Tunes Stability and Folding Kinetics of bcl2Mid G‐Quadruplex

Abstract Cytosine methylation, a key epigenetic modification in the regulation of gene expression, raises intriguing questions about its role in the formation and thermodynamic stability of G‐quadruplex (G4) structures. We investigated the impact of the 5‐methylcytosine residue (C m ) on the well‐characterized bcl2Mid G4 structure that forms in a GC‐rich region of the B‐cell lymphoma 2 (BCL2) gene promoter, which influences its expression. Using solution‐state NMR and biophysical techniques, we discovered an unexpected sequence‐specific effect of C m on the folding kinetics of bcl2Mid G4. Specifically, substituting cytosine at position C6 with C6 m   slows down G4 folding kinetics and influences the equilibrium between major and minor structures in the presence of K + ions. Notably, the increased population of the minor structure enabled the characterization of its previously unidentified topology. Additionally, the presence of a single C m residue induces local structural rearrangements in the major G4 structure and decreases its thermodynamic stability. Furthermore, we found that the zinc finger 3 motif of the Sp1 transcription factor preferentially binds to the minor G4 structure. These results suggest that C m not only influences G4 polymorphism but may also regulate interactions with transcription factors, potentially affecting the regulation of gene expression.