Oxidative probing of the G4 DNA structure induced in double-stranded DNA by molecular crowding or pyridostatin

Major advances have been made recently in the application of the highly selective G4 DNA ligand pyridostatin (PDS) for targeting and visualization of this noncanonical DNA structure in eukaryotic genomes. However, the interaction of PDS with the G4 structure constrained by double-stranded DNA has not yet been analyzed. Here, we induced folding of G4 structures in double-stranded DNA promoter fragments of several oncogenes by annealing the DNA under molecular crowding conditions created by polyethylene glycol (PEG) or in the presence of PDS. Both PEG and PDS induced similar DNA folding, as demonstrated by gel mobility assays and S1 nuclease cleavage. The cationic porphyrin derivative ZnP1 was used to probe the G4 structure in both conditions and thus provided with "footprint" of PDS. The PEG-stabilized G4 structure was susceptible to photo-induced oxidation by ZnP1 and tended to revert to a duplex after oxidation. Guanines in the 5'-tetrad were the most accessible to ZnP1 and became protected from oxidation upon binding of PDS which prevented the G4 structure from rearranging into a double helix. The study demonstrates the applicability of porphyrin ZnP1 for the probing of G4 structures in the genomic context and footprinting of G4 specific ligands.