Electrochemical approach for the analysis of DNA degradation in native DNA and apoptotic cells

Abstract

The aim of this work was to develop an electrochemical approach for the analysis of DNA degradation and fragmentation in apoptotic cells. DNA damage is considered one of the major causes of human diseases. We analyzed the cleavage processes of the circular plasmid pTagGFP2-N and calf thymus DNA, which were exposed to restriction endonucleases (the restriction endonucleases BstMC I and AluB I and the nonspecific endonuclease I). Genomic DNA from the leukemia K562 cell line was used as a marker of the early and late (mature) stages of apoptosis. Registration of direct electrochemical oxidation of nucleobases of DNA molecules subjected to restriction endonuclease or apoptosis processes was proposed for the detection of these biochemical events. Label-free differential pulse voltammetry (DPV) has been used to measure endonuclease activities and DNA damage using carbon nanotube-modified electrodes. The present DPV technique provides a promising platform for high-throughput screening of DNA hydrolases and for registering the efficiency of apoptotic processes. DPV comparative analysis of the circular plasmid pTagGFP2-N in its native supercoiled state and plasmids restricted to 4 and 23 parts revealed significant differences in their electrochemical behavior. Electrochemical analysis was fully confirmed by means of traditional methods of DNA analysis and registration of apoptotic process, such as gel electrophoresis and flow cytometry.

Keywords: Apoptosis; Bioelectrochemistry; Carbon nanotubes; DNA fragmentation; Modified electrodes; Plasmid DNA.

Graphical abstract

Fig. 1

DPVs for SPE/SWCNT in the absence (dashed line, blank electrode) or presence (red line) of dsDNA from herring sperm and (blue line) plasmid DNA at a concentration of 0.1 mg/mL.

Fig. 2

DPVs of the SPE/SWCNTs in the presence (blue line) of the plasmid DNA and (green line) of the plasmid DNA restricted to 4 fragments at a concentration of 0.1 mg/mL in the absence of a DNA blank electrode (dashed line).

Fig. 3

DPVs for the SPE/SWCNT/plasmid DNA restricted into four fragments at different concentrations (0.05–0.1) mg/mL.

Fig. 4

(A) DPVs for the SPE/SWCNT/circular plasmid DNA and plasmid DNA restricted to 4 or 23 fragments at a concentration of 0.01 mg/mL; (B) Derivatives dI/dE of signals for the SPE/SWCNT/circular plasmid DNA or circular plasmid DNA restricted to 4 or 23 fragments; (C) Histograms corresponding to the ratio of the I/C signals for circular plasmid DNA (Pl) or plasmid DNA restricted to 4 (Pl/4) and 23 (Pl/23) fragments at a concentration of 0.01 mg/mL; (D) Agarose gel electrophoresis of pTagGFP2-N subjected to restriction with the specific nucleases BstMC I or AluB I or nonspecific endonuclease I. Original agarose gel image is shown inFig. 1Sin the supplementary file.

Fig. 5

DPVs for SPE/SWCNTs in the absence (dashed line, blank electrode) and presence (red line) of native calf thymus DNA at a concentration of 0.1 mg/mL.

Fig. 6

DPVs for SPE/SWCNTs in the presence of (A) plasmid DNA and (B) calf thymus DNA. The sample is restrictive medium with endonuclease I and DNA (red lines), the control is restrictive medium with endonuclease without DNA (black line), and the blue lines represent only the degraded plasmid and calf thymus DNA as the difference between sample and control. The plasmid DNA and calf thymus DNA concentrations were 0.17 and 0.12 mg/mL, respectively. (C) Agarose gel electrophoresis of calf thymus DNA subjected to restriction with nonspecific endonuclease I. Original agarose gel image is shown inFig. 1Sin the supplementary file.

Fig. 7

DPV profiles for SPE/SWCNTs in the absence (dashed line, blank electrode) or presence (red line) of native calf thymus DNA and (orange line) of intact DNA at a concentration of 0.55 mg/mL.

Fig. 8

Detection of cell apoptosis and DNA degradation in apoptotic cells К562 cells were incubated with l-asparaginase for 60 or 72 h. (A) Histograms of live, apoptotic and dead cells. (B) Representative flow cytometry plots for cells labeled with annexin V–FITC and PI. The ratios of living cells (lower left quadrants), early apoptotic cells (lower right quadrants), late apoptotic cells (upper right quadrants) and dead cells (upper left quadrants) are presented. (C) Histograms showing an increasing number of TUNEL-positive cells, indicating an increase in the percentage of cells with degraded DNA after apoptosis induction. (D) Representative TUNEL assay flow cytometry plots for incubated cells. (E) Agarose gel electrophoresis of DNA isolated from control cells or cells after apoptosis induction. The results are presented as the mean ± standard error of the mean (SEM); *p ≤ 0.05 vs. control untreated cells according to Student's t–test; n = 4. Original agarose gel image is shown inFig. 2Sin the supplementary file.

Fig. 9

Detection of degraded DNA from apoptotic cells via an electrochemical method (A) DPV profiles for SPE/SWCNTs in the absence (dashed line, blank electrode) or presence (red line) of DNA during late apoptosis (blue line) of DNA during early apoptosis and (orange line) of intact DNA at a concentration of 0.55 mg/mL; (B) histograms corresponding to the ratio of the DNA electrooxidation signals at a concentration of 0.55 mg/mL in apoptotic cells at different stages of cell death.