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

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.