Determination of ligand selectivity to G-tetrad through an AMCA fluorescence quenching approach

The selective binding of ligand molecules towards the 5' and 3' ends of G-quadruplex (G4) may differentially affect the physiological function of G4s. However, there is still a lack of sensitive and low-cost approaches to accurately measure the binding preference of ligands on G4s, although multiple ways have been developed to evaluate the interaction between ligands and G4s. Here, we propose a new protocol named G4-AFQ to test the selectivity of ligands towards the two terminal G-tetrads of G4s. In this protocol, the fluorophore AMCA is respectively modified at the 5' or 3' end of G4, and which end of AMCA fluorescence is quenched means that the ligand binds to the G-tetrad at that end. Through G4-AFQ, the affinity constant of ligands towards the binding site can also be obtained. Compared with the commonly used nuclear magnetic resonance (NMR) method, G4-AFQ is more convenient, sensitive, cost-effective, and suitable for the measurement of the vast majority of G4 ligands, with a great potential for widespread application.