Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker.
This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.
Havran
Ludek
L
Institute of Biophysics, v.v.i, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65, Brno, Czech Republic.
Vacek
Jan
J
Cahová
Katerina
K
Fojta
Miroslav
M
D000477:Alkylating Agents / Q000737:chemistry; D015415:Biomarkers / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D002244:Carbon / Q000737:chemistry*; D004249:DNA Damage; D045643:DNA Repair Enzymes / Q000032:analysis* / Q000378:metabolism; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D005090:Exodeoxyribonucleases / Q000378:metabolism; D009992:Osmium / Q000737:chemistry*; D011687:Purines / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013463:Sulfuric Acid Esters / Q000737:chemistry
D000477:Alkylating Agents; D015415:Biomarkers; D011687:Purines; D013463:Sulfuric Acid Esters; D009992:Osmium; D002244:Carbon; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045643:DNA Repair Enzymes; C007482:dimethyl sulfate