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.
Keywords: Circular dichroism; FRET; G-quadruplex; Ligand; PEG.
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