Vpliv epigenetskih modifikacij na strukturne in termodinamske lastnosti DNA

Cytosine methylation is a crucial epigenetic modification that has subsequently been the focus of intense studies, as it plays a significant role in the regulation of gene expression. Despite this considerable attention, its impact on the formation and stability of G-quadruplex structures remains largely unexplored. We deemed it important to focus on the folding of a G-rich sequence and to evaluate its thermodynamic and kinetic aspects as cytosine residues get methylated. In this regard, we studied the impact of introducing a 5-methylcytosine residue into the oligonucleotide sequence 5'-d(GGG CGC GGG AAGGATT GGG C GGG), which folds into the G-quadruplex structure known as bcl2Mid G4. This well-characterized G4 structure forms within a GC-rich region located immediately upstream of the P1 promoter of the B-cell lymphoma 2 (BCL2) gene, which plays a crucial role in regulating its expression. Using solution-state NMR and complementary biophysical techniques, such as UV-Vis, CD spectroscopy and differential scanning calorimetry (DSC), we discovered a previously unknown effect of 5-methylcytosine on G-quadruplex structures. Specifically, the substitution of the cytosine at position C6 with the modified analogue C6$^m$ destabilizes the predominant structure and increases the ratio of the minor G-quadruplex structure. The altered ratio of the major and minor G-quadruplex structures in the equilibrium, coupled to the changes in the rate of DNA (re)folding, highlights an unexpectedly profound effect of a single 5-methylcytosine residue on the folding and thermodynamic stabilities of guanine-rich DNA. Furthermore, by investigating the details of the minor species resolved upon introducing the 5-methylcytosine modification, we obtained insights into a previously unexplored G-quadruplex structure that exhibits an intriguing snapback element at its 5'-end. Additionally, our findings indicate that the zinc finger 3 motif, which is a part of the DNA-binding domain of the Sp1 transcription factor, preferentially binds to the minor G-quadruplex structure, while it does not show specific interactions with the major G-quadruplex structure. Our findings demonstrate that the seemingly modest impacts of 5-methylcytosine modification(s) on the polymorphism of G-quadruplex structures may be of fundamental importance in contexts of modulating DNA interactions with transcription factors and, furthermore, the regulation of gene expression.