title: Assessment of selectivity of G-quadruplex ligands via an optimised FRET melting assay.
abstract: Four-stranded G-quadruplex (G4) DNA structures are promising drug targets as these non-canonical structures appear to regulate gene expression and telomere growth. Although all types of G4 are stabilised by quartets of four guanosines, differences in loops, grooves, and flanking bases, result in an impressive structural diversity among G4 structures that may allow selective recognition by small molecule ligands. We adapted the previously described Förster resonance energy transfer (FRET) melting assay to evaluate the selectivity of G4 ligands for different G-quadruplex topologies. We demonstrated that the incorporation of FAM and Tamra fluorescent dyes and the presence of PEG influenced the structures adopted by certain sequences with G-quadruplex-forming potential. Optimisation of the measurement conditions ensured the folding and thermal stability of a selected set of G4 DNA oligonucleotides in a measurable temperature range with and without ligand. The optimised method enabled comparison of well known G4 ligands such as TmPyP4, Braco19, pyridostatin, 360A, PhenDC3, and TrisQ.
authors.affiliation: Univ. Bordeaux, ARNA Laboratory, F-33000 Bordeaux, France; INSERM U869, IECB, F-33600 Pessac, France.
authors.forename: Aurore
authors.initials: A
authors.lastname: De Rache
authors.affiliation: Univ. Bordeaux, ARNA Laboratory, F-33000 Bordeaux, France; INSERM U869, IECB, F-33600 Pessac, France. Electronic address: jean-louis.mergny@inserm.fr.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Mergny
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D054856:G-Quadruplexes / Q000187:drug effects; D006801:Humans; D008024:Ligands; D009691:Nucleic Acid Denaturation; D011092:Polyethylene Glycols / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D011092:Polyethylene Glycols; D004247:DNA; D011188:Potassium
keywords: Circular dichroism; FRET; G-quadruplex; Ligand; PEG