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Ligands of G-quadruplex have received great attention because of their potential anticancer activity. Here, we designed a label-free electrochemical method to identify electrochemically active molecules that stabilize G-quadruplexes in homogeneous medium. Daunorubicin (DNR) was used as model drug. In the presence of GDNA, the voltammetry of DNR changed. The binding constant (β) and binding-site number (m) of GDNA–mDNR are simultaneously obtained from dependence of the current on the amount of added GDNA in voltammetry, concluding that β=(2.37±2.25)×107 M−1and m was near 1, respectively. Control experiment was performed, indicating that this method can specific identify G-quadruplex-binding ligand. The interaction mechanism of DNR with GDNA was also investigated by circular dichroism (CD). The result demonstrated that it is a simple and selective method for identification electroactive ligand with potential anticancer activity. Keywords: G-quadruplex, Ligand, Label-free, Electroactive

The G-rich overhang of human telomere tends to form a G-quadruplex structure, and G-quadruplex formation can effectively inhibit telomerase activity in most cancer cells. Therefore, it is important to identify the formation and properties of the G-quadruplex, with the particular aim of selecting G-quadruplex-binding ligands that could potentially lead to the development of anticancer therapeutic agents. With this goal in mind, we report a fluorescence resonance energy transfer (FRET) assay system for the identification of G-quadruplex ligands using DNA-functionalized gold nanoparticles (DNA-GNPs) as the fluorescence quencher and a carboxyfluorescein (FAM)-tagged human telomeric sequence (F-GDNA) as the recognition probe. A thiolated complementary strand of human telomeric DNA (cDNA), which first adheres to the surface of the GNPs and then hybridizes with F-GDNA, results in the fluorescence quenching of F-GDNA by the GNPs. However, fluorescence is restored when single-stranded F-GDNA folds into a G-quadruplex structure upon the binding of quadruplex ligands, leading to the release of F-GDNA from the surface of the GNPs. Combined data from fluorescence measurements and CD spectroscopy indicated that ligands selected by this FRET method could induce GDNA to form a G-quadruplex. Therefore, this FRET G-quadruplex assay is a simple and effective approach to identify quadruplex-binding ligands, and, as such, it promises to provide a solid foundation for the development of novel anticancer therapeutic agents.

Ligands that bind and stabilize G-quadruplex DNA structures are of significant interest because of their potential to inhibit telomerase and halt tumor cell proliferation. An electrochemical method was proposed to select natural G-quadruplex-binding ligands using ferrocene as the electrochemical indicator.