Figure 1.
Interaction of 6MP with CT-DNA.
UV-visible absorption spectra of 6MP (25 μM) in presence of increasing concentrations of CT-DNA in Tris-HCl buffer (pH 7.2). Hyperchromism was observed with increasing concentration of CT-DNA confirming the interaction of 6MP and DNA. Structure of 6 Mercaptopurine is shown in the inset.
Figure 2.
Interaction of 6MP with CT-DNA studied using fluorescence spectroscopy.
Fluorescence emission spectra of 6MP (50 μM) in the presence of increasing concentrations of CT-DNA. Increase in the fluorescent intensity was observed with increasing DNA concentration. Excitation wavelength was 280 nm and emission was recorded as shown in figure.
Figure 3.
Stern-Volmer plot for interaction of 6MP with CT-DNA.
The fluorescent intensity was found to be directly proportional to DNA concentration. Binding constant of 7.8×103M−1 was obtained from the slope.
Figure 4.
Competitive displacement assays.
(A) Fluorescence titration of CT-DNA and EB (intercalator) complex with 6MP. EB-DNA complex was excited at 471 nm and emission spectra were recorded from 500–700 nm. No change in fluorescence intensity was recorded with addition of increasing concentration of 6MP (B) Fluorescence titration of CT-DNA and AO (intercalator) complex with 6MP. CT-DNA-AO complex was excited at 471 nm and emission spectra were recorded from 500–600 nm. No change in fluorescence intensity was recorded with addition of increasing concentration of 6MP. (C) Fluorescence titration of CT- DNA and Hoechst (groove binder) complex with 6MP. Fluorescence intensity decreases with subsequent addition of 6MP. CT-DNA-Hoechst complex was excited at 343 nm and emission spectra were recorded from 360–600 nm.
Figure 5.
Stern-Volmer plot for fluorescence quenching of 6MP (50 μM) by KI in absence and presence of CT-DNA (100 μM). Quenching of 6MP fluorescent intensity was done using KI in absence and presence of CT-DNA and quenching constant was calculated in both the case. Difference in Ksv value was further used to investigate the binding mode of 6MP and DNA.
Figure 6.
To study the role of electrostatic effect on 6MP-DNA binding, NaCl was used. Maximum emission intensity plot of 6MP-DNA was plotted with increasing concentration of NaCl (0–70mM). Excitation wavelength was 280 nm. Increase in fluorescence intensity suggests for a possible electrostatic interaction between 6MP and CT-DNA.
Figure 7.
Effect of 6MP on CD spectra of CT-DNA.
CD spectra of CT-DNA (30 μM) in 10mM Tris-HCl (pH 7.2) with the addition of varying concentration of 6MP. Each spectrum was obtained at 25°C with a 10 mm path length cell.
Figure 8.
Effect of increasing the concentration of 6MP on the viscosity of CT-DNA.
The concentration of CT-DNA was kept constant (100 μM) with increasing amount of 6MP. Values reported are mean of three independent experiments.
Figure 9.
Molecular docked structure of 6MP complex with DNA.
Dodecamer duplex sequence (CGCGAATTCGCG)2 (PDB ID: 1BNA) was used in the docking studies. The binding energy of the complex system was found to be −116.97 kJ/mole.
Figure 10.
6MP induced generation of superoxide anion.
Concentration dependent photo generation of superoxide anion by 6MP. Indicated concentration of 6MP was exposed to white light for 1h at RT and absorbance was measured at 560±SEM of three independent experiments. *p value <0.01 when compared to control.
Figure 11.
6MP induced damage to plasmid DNA in presence of light.
Agarose gel electrophoresis pattern of ethidium bromide stained pBR322 DNA after the treatment with 6MP in presence of white light. Lane ‘A’ depicts the ‘Control’ which contain only plasmid DNA. The concentrations of 6MP in lanes ‘B–F’ was 100, 200, 300, 400 and 500 μM respectively. Arrows indicating OC and SC on the right represent the open circular and supercoiled forms of plasmid DNA.
Figure 12.
6MP induced human lymphocyte DNA breakage.
Comet tail length obtained after treatment with 6MP in dark and light. Values reported are ±SEM of three independent experiments. *p value <0.01 when compared to control.