Figure 5.

cPNA2 invasion studies. (A) Chemical probing of plasmid with BCL2 insert incubated with cPNA2 only and with both cPNA2 and bis-PNA. Incubation of plasmids with PNAs was performed in 20 mM KCl (pH 7.4) at 37°C overnight with molar ratio 100:1 PNA to DNA. Control sample was incubated only in TE buffer. Samples were chemically modified to a total volume of 50 μl with 2.5 mM OsO₄ plus 2.5 mM 2.2′-dipyridyl disulfide for 5 min at room temperature, 2 μl DEPC for 5 min at room temperature, 2 μl 10% DMS in ethanol for 15 min at 15°C. Plasmids with two BCL2 insert were tested: with original quadruplex forming BCL2 sequence and with mutant BCL2 sequence not forming quadruplex. G-rich strand was analyzed. (B) A1 adenine modification. Intensity of the band corresponding to A1 adenine was normalized on the intensity of whole lane with background subtraction and plotted. (C) DMS modification profiles for the plasmid with original BCL2 insert incubated alone or in the presence of cPNA2 and bis-PNA. The two guanines outside of the G-quadruplex-forming region are underlined.