Electrochemical sensing of DNA damage Miroslav Fojta Olsztyn-Lańsk, September 20th, 2007 Institute of Biophysics Department of Biophysical Chemistry and Molecular Oncology Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis DNA damage Elektrochemické metody … 1922 Jaroslav Heyrovský: polarografie 1959 Nobelova cena základ celé škály široce využívaných elektrochemických metod 29 DIA3 Elektrochemické metody … -I -E Xm-n Xm n e- identifikace elektrochemicky aktivní látky (co to je) kvantifikace (jaká je koncentrace) kapající rtuťová elektroda Elektrochemické metody … -I -E Xm Xm-n n e- •visicí rtuťová kapková elektroda •pevné elektrody Xm Xm-n n e- tn_Emil late 1950s, Emil Paleček: DNA polarography nature1 nature2 DSCF0004 potenciál/V vs. SCE -2.0 -1.0 +1.0 - + G Gox Aox CA cytosine + adenine reduction oxidation guanine adenine mercury or amalgam electrodes (primarily) carbon elektrodes 7,8 –dihydro guanine formation DNA is electrochemically active potential/V vs. SCE -2.0 -1.0 +1.0 - + G Gox Aox CA cytosine + adenine guanine adenine bases sugar-phosphate backbone 3 1 2 mercury or amalgam electrodes (primarily) carbon electrodes ADSORPTION/DESORPTION distorted double-helix DNA is electrochemically active early studies by polarography: damage to DNA can be detected ioniz strand breaks UV distortions due to base damage single stranded regions in dsDNA etc. transfer transfer adsorption Adsorptive Transfer Stripping Øinstead of ~milliliter volumes, several microliters are sufficient for analysis Øanalysis of reaction mixtures with substances that interfere in „conventiona“ voltammetry (including DNA damaging agents) • • DNA-modified electrode = a simple electrochemical sensor for DNA damage •electrode = signal transducer •„recognition layer“ of DNA at its surface exposure to the analyzed sample DNA modified electrode washing&transfer into electrochemical cell detection (signal mesurement) interaction with the damaging substance DNA-modified electrode = a simple electrochemical sensor for DNA damage chemical modification of DNA can: •cause strand breakage detectable primarily with mercury (amalgam) electrodes •cause distotions of the double helix detectable primarily with mercury (amalgam) electrodes •hit electroactive sites of nucleobases thus affecting their electrochemical activity (mercury or carbon electrodes) •result in introducing new electroactive moieties (principially any electrode - depending on the electroactive group introduced) Detecting strand breaks with mercury-based electrodes difference in behavior of covalently closed circular and nicked or linear DNAs at a mercury electrode surface denaturation of dsDNA at the HMDE within the „region U“ slow scan from positive to negative potentials dsDNA unwinding takes place before the potential of the ssDNA-specific peak 3 is reached DNA with free ends (breaks) extensive unwinding of scDNA not possible surface denaturation of dsDNA at the HMDE within the „region U“ High sensitivity of ssb detection with mercury electrodes •one break in ~1% of a ~3 kbp plasmid molecules can be detected •that is one lesion among ~2x105 intact nucleotides •200 ng of DNA per analysis (better sensitivity than agarose electrophoresis) •detection of multiple strand breaks in one molecule possible (not possible by means of native electrophoresis) guanine oxidation signal at carbon electrodes is not sensitive to formation of individual strand breaks • practically indistinguishable responses of sc, oc and linear DNAs • small sensitivity to DNA structure: intact dsDNA yields a large signal • absence of (extensive) surface denaturation of dsDNA at carbon • Gox Gox cleavage of scDNA by DNase I: HMDE, peak 3 PGE, peak Gox Mercury electrode modified with scDNA: sensor for DNA damaging agents scDNA ·OH ocDNA ACV E/V -0.8 -1.6 1 E/V -0.8 -1.6 3 1 ACV reducing agent (ascorbate) example of the sensor application: detection DNA damaging agents in waste (industrial) waters (uranium mines, Dolní Rožínka) mine water – input of purification plant output of the water purification plant blank (containing considerable amounts of transition metals like Fe, Mn) working with „dangerous“ mercury should be avoided? • similar responses to DNA damage like with the HMDE can be obtained • •with mercury film electrodes (Kubičárová 2000) • • • • •with amalgam electrodes (Cahová-Kuchaříková, Fadrná, Yosypchuk, Novotný 2004) • AC voltammograms of sc, linear ds and denatured DNA at m-AgSAE changes in the peak 3 height (at m-AgSAE) due to scDNA exposure to a chemical nuclease Cu(phen)2 Cu(phen)2 studies of cleavage of DNA at the electrode surface by electrochemically generated reactive species e.g., hydroxyl radicals (or other ROS) can be generated via electrochemically controlled Fentonovy/Haber-Weissovy reactions scDNA-modified electrode was dipped in solution containing Fe/EDTA and H2O2 (neor O2) and potential (Ec) ensuring redox cycling of the metal is applied for certain time then, DNA response is measured with the same electrode (a) EC = 0 V; (b) EC = 0.2 V; (c) EC = 0.4 V applied for 60 s Peak 3 intensity (=the amount of SB, degree of DNA damage) depends on the potetial applied: Fe2+ Fe3+ Fe3+ + e- Fe2+ Fe2+ + H2O2 .OH + OH- + Fe3+ O2 + e- + 2H+ H2O2 if the potential Ec is sufficiently negative for iron reduction [from Fe(III) to Fe(II)], redox cycling is maintained, hydroxyl radicals are produced and DNA is nicked phen+Cu+O2 Cu+O2 in the presence of 1,10-phenanthroline, a ligand stabilizing Cu(I), stronger DNA damaging effect was observed at more negative potentials •analogous effects were observed in the presence of copper (and O2) • •in this case efficient DNA cleavage is observed only in a narrow potential region where Cu(I) ions (stabilized by coordination with DNA bases) can mediate ROS formation Cu+O2 Cu2+ Cu0 Cu+(DNA) O2 Cu přidat chrom oxygen not required Cr(III) potentiates DNA damage in the presence of oxygen (Electroanal., in press) ? Cr(II) Cr(III) (electrochemically) electrochemically probably not Detection of DNA degradation with carbon electrodes carbon electrode intercalator accumulated in the DNA „sensitive“ layer yields a voltammetric peak due to degradation of the DNA layer, less intercalator in boud and its signal is decreased signal decrease due to DNA degradation by Cu(phen)2 intercalator Redox indicator based technique (Labuda et al.) : •the indicator can recognize intact DNA from (extensively) damaged DNA uhlíková elektroda i intercalator application: testing of antioxidant capacity of different substances •DNA degraded by hydroxyl radicals •antioxidants counteract the hydroxyl radicals effects Redox indicator based technique (Labuda et al.) : •the indicator can recognize intact DNA from (extensively) damaged DNA Damage to DNA bases E/V 0.4 1.2 peak Gox •techniques based on a loss of electrochemical activity of chemically modified bases •usually guanine •guanine signals at carbon or mercury electrodes • •alkylating agents, hydrazines, PCBs, cytostatics, acridines, arsenic oxide… • E/V 0.4 1.2 peak Gox •some base adducts yield electrochemical signals distict from those corresponding to the unaffected bases •e.g., 8-oxoguanine • mixture of G a 8-oxoG 8-oxoG elecrochemically generated in DNA at GCE in the presence of adriamycin (A.M. Oliveira-Brett) cisplatin LMBP-obr1 cisplatin modifies primarily guanines Gox Aox Gox Aox high cis-platination levels: diminution of peak Gox at carbon (cisplatin/nucleotide ratio rb=1.0, time dependence) for rb < 0.1 no reliable changes in peak Gox intensity under the same conditons 0 1h 3 h cisplatin unmodified DNA DNA-Os,bipy free Os,bipy DNA modified with osmium tetroxide complexes •not „classical“ DNA damaging agents •chemical probes of DNA structure • •indirect technique of DNA damage detection Ru3+ G •utilizes changes of accessibility of guanine bases for interaction with a redox mediator upon DNA damage •during diffusion through the heme protein layer, the substance is „metabolically activated“ • •DNA adduct is formed • •due to the adduct, the double helix is „unravelled“ making neighboring bases (guanines) more accessible for Ru-mediated oxidation • SIGNAL INCREASES Sensor for (geno)toxicity testing (Rusling et al.) STYRENE TOLUENE (not „activated“ by the heme enzymes) Sensor for (geno)toxicity testing (Rusling et al.) base damage repair endonuclease break scDNA scDNA ocDNA 3 1 1 1 base damage converted to strand breaks → sensitive detection at mecury or amalgam electrodes a – intact scDNA b –endoV treated scDNA c – UV irradiated scDNA d – UV+endoV dependence on UV dose dependence on enzymatic cleavage time irradiated non irradiated irradiated, peak Gox at carbon Py dimers detected by endonuclease V scDNA scDNA +ExoIII scDNA +DMS scDNA +DMS +exoIII (peak 3 details) apurinic sites detected by exonuclease III UV endo V break exo III scDNA scDNA ocDNA ssDNA 3 1 1 1 1 3 enhancement of the ssb signal using exonuclease III cleavage substrate specificity of the enzymes → specificity of adduct detection Utilization of an electroactive marker in detection of DNA damage (OsO4,bipy) Øcommercially available chromosomal (=linear) DNAs (such as calf thymus or salmon sperm DNA) produce a considerable peak 3 Øonly small relative changes due to additonal damage (depending on the sample quality) DMS exo III „dose“ dependence (conc. of DMS) Os,bipy undamaged damaged signals of the marker (at carbon):