Guanine-rich oligonucleotides (GROs) can fold into G-quadruplex (G4) structures. The diverse roles of G4 structures, particularly as targets for drug design, anticancer agents, and drug delivery systems, highlight their critical significance in cancer research. However, the formation of G4 structures is highly dependent on the specific nucleotide sequences and the number of G-tracts within each GRO. In vitro studies using circular dichroism (CD), nuclear magnetic resonance (NMR), and polyacrylamide gel electrophoresis (PAGE) demonstrated that GROs with fewer than four G-tracts can form intermolecular G4 structures in K