Figure 6.

Evaluation of BG4 binding on a plasmid containing telomeric DNA sequences that have the ability to fold into G-quadruplexes. (A) pSXneo135(T2AG3) and pSXneo270(T2AG3) contain 0.8  and 1.6 kb long telomeric repeats [(T₂AG₃)_n], respectively and is shown to have the ability to form G-quadruplexes.⁵⁴ pSV4 contains 1.19 kb long region containing random sequence, devoid of any non-B DNA motifs. (B) CD spectra recorded for pSXneo135, pSXneo270 and pSV4 in presence and absence of 100 mM KCl, at room temperature, within wavelength range of 200–300 nm (10 cycles), using a spectropolarimeter (JASCO J-810) at a scan speed of 50 nm/min. Absorption measured for buffer alone (15 cycles) was subtracted from each sample and the resulting spectra was plotted. (C) Comparison of BG4-binding efficiency between pSXneo135 (lanes 1–4), pSXneo270 (lanes 5–8) and pSV4 (lanes 9–12) as determined by shift in mobility of supercoiled form of the plasmid, indicated using dotted box. In each case, plasmid DNA was incubated with 100 mM KCl at 37°C for 1 h, followed by BG4 (0, 1.5, 2.3, 3 µg) at 4°C for 1 h, and resolved on 1.2% agarose gel containing 25 mM KCl at 100 V, at room temperature. BG4-bound supercoiled forms of pSXneo135 shift substantially (lanes 1–4) as do those of pSXneo270 (lanes 5–8), unlike the control plasmid pSV4 (lanes 9–12), when electrophoresed for longer time periods (5 h compared to 3 h). (D) Bar diagram showing quantification of shift in the mobility of supercoiled plasmids upon addition of BG4 (0.00, 1.5, 2.3, 3 µg). Y-axis denotes the mobility shift of the supercoiled form.