Highly Specific Inhibition of c-Myc Oncogene Expression through Shifting the G-Quadruplex toward a Triplex Structure

G-quadruplexes act as transcriptional triggers in oncogenes, and small molecules targeting them show anticancer potential but lack sequence specificity, risking off-target effects. Here, we introduce original fold-back triplex-forming oligonucleotides (FTFOs) as precise alternatives. Physicochemical and biological analyses revealed that FTFOs unfold the c-Myc G-quadruplex and repress oncogene regulatory element by forming noncanonical triplexes that block G-quadruplex DNA from interacting with transcription factors. Derived design rules enabled the identification of FTFOs that reduce c-Myc expression in the Del4 plasmid by up to 80%, significantly impairing cancer cell growth, confluence, mobility, and endogenous c-Myc expression. This work presents FTFOs as an innovative sequence-specific strategy for arresting G-quadruplex structures via triplex formation and suppressing oncogene activity, offering a promising avenue for targeted anticancer therapy.