Figure 5.

DMS probing of BG4-bound G-quadruplex DNA substrates. (A) DMS protection assay of RT17 (HIF1α) in presence or absence of KCl and BG4 antibody. Since G-quadruplex structure does not form in the absence of KCl on RT17, all guanines react to DMS resulting in specific bands (lane 1, guanine residues marked in grey). Addition of potassium stabilizes the G-quadruplex DNA, and those G residues are protected and indicated in brown (lane 2). BG4 binding shields the sequence from DMS leading to additional protection and footprint (lane 3, protected guanine residues are indicated in blue). RT17 was incubated at 37°C in presence of KCl, and then incubated with BG4. DMS probing was performed at 37°C on both samples, followed by piperidine cleavage and purification. The reaction products were resolved on 18% denaturing PAGE. Brown ovals indicate the guanine residues protected in presence of potassium and in the absence of BG4. Blue ovals indicate the protected residues in presence of both potassium and BG4. (B, C) DMS probing of KD49 (VEGF) and KD83 (C9ORF72) in presence of KCl and BG4 antibody. BG4 antibody was added (lane 2) to potassium-stabilized G4 (lane 1) and subjected to DMS protection assay. Guanine residues are marked and those protected are indicated in green.