'The role of a G-quadruplex motif in SIRT-1 regulation'

G-quadruplexes are non-canonical secondary structures that form within guanine-rich regions of nucleic acids. These distinctive configurations have been shown to influence several essential cellular processes, including DNA replication, transcription, and RNA translation (1). Many gene promoter regions and 5’ untranslated regions (5’UTRs) are enriched with potential G-quadruplex- forming sequences, which can act as regulatory elements. Notably, these structures can be selectively stabilized by small-molecule ligands, some of which possess well-documented anti- cancer properties (1). SIRT1 is a member of the sirtuin family, known for its role in promoting cell survival and protecting cells from senescence induction (2,3). Using the G-quadruplex prediction tool QGRS Mapper, we identified a guanine-rich sequence within the SIRT1 promoter, located 47 nucleotides upstream of the transcription start site, that is capable of forming multiple G- quadruplex conformations. In previous work carried out in our laboratory, two putative quadruplex- forming sequences (PQS), named L171 and L719, were analyzed. Both sequences demonstrated the ability to fold into stable G-quadruplex structures. Furthermore, polymerase stop assays showed that both PQS were recognized and stabilized by the G-quadruplex ligand Pol3. Additionally, FRET experiments confirmed that both Pol3 and Emicoron—another well- characterized G-quadruplex ligand with established anti-cancer activity (4,5) are capable of binding to the PQS within the SIRT1 promoter. Based on these findings, we treated MCF-7 breast cancer cells with these ligands and observed a significant decrease in both SIRT1 mRNA and protein levels. This downregulation induced a senescent phenotype in the cells, as evidenced by an increase in the expression of P21, a key marker of senescence. Finally, we demonstrated that treatment with these G-quadruplex-binding compounds significantly reduced the viability of MCF-7 cells, as assessed by MTT and crystal violet (CV) assays. These results support the potential use of G-quadruplex-stabilizing ligands as promising therapeutic agents in cancer treatment.