Development of a fluorescent intercalator displacement assay (G4-FID) for establishing quadruplex-DNA affinity and selectivity of putative ligands

A fluorescent intercalator displacement assay (G4-FID) has been designed based on the displacement of thiazole orange (TO) positioned onto a quadruplex-forming oligonucleotide by putative ligands. This technique was validated by the use of a set of representative and fully characterized G-quadruplex binders (ranging from pyridodicarboxamide to macrocyclic ligands). To further extend its applicability, a comparative version has been developed which allows a rapid and viable determination of quadruplex- over duplex-selectivity.