S elektrodami na biomakromolekuly: uplatnění elektrochemických metod v moderní biochemii, molekulární biologii a biomedicíně doc. RNDr. Miroslav Fojta, CSc. MU, 4. května 2010 nature1 50-letá výročí nature2 Nobelova cena za polarografii (Jaroslav Heyrovský) Elektrochemická aktivita DNA (Emil Paleček) Elektrochemické metody … polarografie -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- Elektrochemické metody … voltametrie Jak TOHLE souvisí s DNA, bílkovinami, experimentální biologií, biomedicínou? •víte, co se děje s elektrony v molekulách luminoforů, když měříte fluorescenci (nebo „jen“ fotíte gel značený ethidiem)? Víte, co to je molekulový p-orbital? Víte, co se děje na CCD detektoru? •víte, proč nemáte nanášet na PAGE vzorky DNA s velkou koncentrací solí, jinak dostanete „hnusný“ gel? •víte, proč glycerol indukuje hvězdičkovou aktivitu restriktáz? •atd. •pořád (fyzikální) chemie, pořád fyzika!!! • • • • elektrochemie proteinů Elektrochemie proteinů od dvacátých let XX. století (J. Heyrovský, R. Brdička) brdicka1 +1 -1 -2 E/V -I W Y elektrochemická oxidace tryptofanu a tyrozinu signály peptidů a proteinů obsahujících cystein (cystin) UHLÍKOVÉ ELEKTRODY RTUŤOVÉ ELEKTRODY redukce vazby S-Hg redukce vazby S-S (cystin) Brdičkova reakce (v přítomnosti Co) katalytické vylučování vodíku prenatriová vlna pík H Brdičkova reakce Brdička, 1933: polarografické vlny v přítomnosti sérových proteinů a solí kobaltu Brdičkova reakce a diagnostika rakoviny -kdysi poměrně rozšířený diagnostický test -dnes renesance tohoto přístupu -R. Kizek a spol (MENDELU) -rakovina a metalothioneiny Stanovení fytochelatinů v rostlinných buňkách Fytochelatiny: „rostlinné metalothioneiny“ •detoxifikace těžkých kovů v rostlinných buňkách • • • •syntéza indukována těžkými kovy (kadmium) Kadmium navozuje u buněk TBY-2 apoptózu (programovanou buněčnou smrt) M. Fojtova, and A. Kovarik, Genotoxic effect of cadmium is associated with apoptotic changes in tobacco cells, Plant Cell Environ. 23 (2000) 531-537. M. Fojtova, J. Fulneckova, J. Fajkus, and A. Kovarik, Recovery of tobacco cells from cadmium stress is accompanied by DNA repair and increased telomerase activity, J. Exp. Bot. 53 (2002) 2151-2158. TBY-2 TBY-2 po 5 dnech v 50 mM CdSO4 !FIG !FIG ztráta viability fragmentace DNA Øs 10 mM kadmiem si buňky „poradí“ Ø100 mM kadmium indukuje apoptózu dříve než 50 mM kadmium !GRAFFDA Co se děje s hladinou PC v průběhu těchto změn? Jak souvisí s apopotózou/přežitím? Dokážeme PC v extraktech z buněk jednoduše stanovit elektrochemicky? na základě poměru intenzit jednotlivých signálů (tvaru voltamogramu) lze PC odlišit od CG, gEC i od glutathionu (Dorčák a Šestáková, 2005) voltametrie v přítomnosti solí kobaltu (tzv. Brdičkova reakce) Cd2+ TBY-2 kultivace buněk homogenizace centrifugace supernatant naředění na jednotnou koncentraci celkového proteinu adsorpce na rtuťovou elektrodu měření PSA-srovn-2D50-2DK a b Co Co3/2 blank (nic adsorbováno na HMDE) kontrolní buňky (2 dny, bez kadmia) 2 dny, 50 mM CdSO4 a b Co Výška píku „a“ reprodukovatelně reaguje na přítomnosti kadmia v médiu, a to jak v závislosti na jeho koncentraci, tak na době kultivace blank kontrola 10 mM CdSO4 50 mM CdSO4 100 mM CdSO4 Monitorování hladiny fytochelatinů v rostlinných buňkách pomocí elektrochemických metod 4.10. 2006 Srní, 4. metodické dny Biofyzikální ústav AVČR, Brno Laboratoř biofyzikální chemie a molekulární onkologie Centrum biofyzikální chemie, bioelektrochemie a bioanalýzy P1010021 ? Dotaz od jednoho posluchače po přednášce: TO SE JEŠTĚ DĚLÁ?? Já myslel, že to je už k vidění jenom v muzeu… A co DNA? Struktura DNA… 1953: James Watson, Francis Crick, Rosalind Franklin, Maurice Wilkins: dvoušroubovice DNA 1962: Nobelova cena (JW, FC, MW) vysvětlení základních principů uchování, předávání a exprese dědičné informace main_watson_crick Click for larger picture! Click for larger picture! purinové báze thymin (T) pyrimidinové báze cytosin (C) adenin (A) guanin (G) dvoušroubovice 2-deoxyribóza fosfát D:\dokumenty\OPVK\do přednášky\514px-DNA_chemical_structure.svg.png párování bazí v řetězcích DNA tn_Emil 1958 – 1960 Emil Paleček: polarografie DNA nature1 nature2 DSCF0004 Effects of DNA structure on its electrochemical behavior. Detecting DNA damage. Institute of Biophysics Department of Biophysical Chemistry and Molecular Oncology Centre of Biophysical Chemistry, Bioelectrochemistry and Bioanalysis nucleic acids are electroactive •at mercury electrodes, bases A,C and G undergo redox processes • •at carbon electrodes, nucleobases can be oxidized Reduction DNA signals at the mercury electrodes are strongly influenced by DNA structure •this is due to location of the A and C electroactive sites within the Watson-Crick hydrogen bonding system Reduction DNA signals at the mercury electrodes are strongly influenced by DNA structure square-wave voltammetry DNA oxidation at carbon electrodes is less influenced by DNA structure •oxidation sites of guanine and adenine in dsDNA are located closer to the double helix surface and are accessible via the double helix grooves DNA oxidation at carbon electrodes is less influenced by DNA structure At mercury electrodes in weakly alkaline media, adsorption-desorption (tensammetric) signals of nucleic acids can be detected (e.g., using AC polarography, voltammetry, AC Z) •depending on the conditions and on DNA structure, individual components of the polynucleotide chains may be involved in adsorption/desorption processes background electrolyte -at moderate ionic strenght, double-stranded DNA yields peak 1 due to desorption/reorientation of DNA segments adsorbed via the sugar-phosphate backbone 1 double-helix -distorted or regions of double-stranded DNA yield peak 2 double-helix 2 background electrolyte 2 background electrolyte single-stranded (denatured) DNA yields peak 1 (due to the sugar-phosphate backbone) and peak 3 due to desorption/reorientation of DNA segments adsorbed via freely accessible bases 3 5 adsorption/desorption behavior of DNA at electrodes is strongly related to negative charge of its sugar-phosphate backbone (together with a strong adsorption of nucleobases via hydrophobic forces) peptide nucleic acid: DNA analogue with neutral backbone DNA PNA (decamers, identical base sequence) AC Z at HMDE differential pulse polarography •used in nucleic acid studies in the 60-70´s •discrimination between ss and dsDNA differential pulse polarography •peak II: high sensitivity to subtle changes of dsDNA structure (&dynamics) • •DNA premelting PREMELT DPP peak II A260 differential pulse polarography •strand breaks Ø ioniz differential pulse polarography Øchemical modification of DNA: platinum adducts Ø distinction of the kind of structural change caused by modification with different Pt complexes peak II: conformation distortion, base pairing preserved peak III: base unpairing (Brabec et al.) Changes of DNA structure at electrically charged surface HMDE 1 3 2 -1.5 -0.5 E (V) DME (SMDE) 1 2 -1.5 -0.5 E (V) 3 U intensities of ssDNA-specific signals prolonged exposure to (accumulation at) potential given on x-axis pH close to neutral (bases not ionized): region T: – negligible structural changes due to adsorption region U: surface denaturation dsDNA dsDNA ssDNA positive or neutral base sugar phosphate • close to the duplex ends (or single-strand breaks), some bases can be unpaired and make contact with the mercury surface • phosphates repelled from negatively charged surface • randomly adsorbed bases represent relatively firm anchor sites • constraints in the double helix cause its (slow) unwinding • more (unpaired) bases are coming into contact with the electrode (in real situation the strands must rotate around one another; the process requires repeated adsorption/desorption events) effects of initial potential and scan direction DNA with or without ends detection of DNA strand breaks using supercoiled DNA and mercury electrodes supercoiled open (nicked) circular linear 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“ detecting DNA damage DNA in the cells is permanently exposed to various chemical or physical agents Ø endogenous - products and intermediates of metabolism Ø exogenous - environmental (radiation, pollutants) Scharer, O. D. (2003) Chemistry and biology of DNA repair, Angew. Chem. Int. Ed. 42, 2946-74. single-strand break double-strand break interruptions of DNA sugar-phosphate backbone abasic sites interruption of the N-glykosidic linkage Øreactive oxygen species Øaction of nucleases Øconsequence of base damage Ø Øspontaneous hydrolysis (depurination) Øconsequence of base damage Ø Most frequent products of DNA damage („lesions“) guanin adenin cytosin thymin base damage: chemical modifications Øalkylation Øoxidative damage Ødeamination Ødamage by UV radiation (sunlight) Ømetabilically activated carcinogens Øanticancer drugs Ø Ø Ø Most frequent products of DNA damage („lesions“) How to detect DNA damage? 1.Techniques involving complete DNA hydrolysis followed by determination of damaged entities by chromatography or mass spectrometry 2. 2.Monitoring of changes in whole (unhydrolyzed) DNA molecules: electrophoretic and immunochemical techniques Can electrochemistry help? 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 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) 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) • 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 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 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) What to do when the DNA damage product of interest: •is not electroactive •does not affect intrinsic electroactive sites of DNA •is too rare to be detectable via e.g. degrease of the guanine signal? base damage repair endonuclease break scDNA scDNA ocDNA 3 1 1 1 base damage converted to strand breaks → sensitive detection at mercury (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 Ligation (repair) of strand breaks Ligatable and unligatable strand breaks only 3‘-OH, 5‘-phospho junctions can be sealed directly by ligases Sensitive detection of strand breaks (DNA damage convertible to strand breaks) with carbon electrodes: should using „bad dangerous“ mercury be avoided? Utilization of an DNA structure-selective electroactive marker •fast reaction with thymine, slow with cytosine, practically no with purines •DNA structure selectivity: thymines in duplex DNA are protected from modification • thymine HMDE voltammetric responses of the Os labels PGE Label-free AC voltammetry at mercury electrode: discrimination between dsDNA without breaks and with breaks Os,bipy: highly selective for ssDNA – strong voltammetric signal due to ssDNA modification sc, oc (ds) ss Creation of ssDNA stretches in dsDNA possessing free ends (=breaks) using exonuclease III (no cleavage of intact DNA) exo III, then Os,bipy; SWV measurement time of enzymatic treatment reactive to Os,bipy nhighly selective for DNA containing breaks or abasic sites (=damaged) nin combination with other DNA repair enzymes, also for other types of nucleobase damage nit is the combination of the DNA repair enzyme combined with the chemical probe Os,bipy, not the carbon electrode, what renders the assay highly sensitive! relative increase of signals: (mixtures intact/single-nicked plasmid DNA, exo III treatment) % of single-nicked DNA in intact scDNA (1 % corresponds to one strand break per 3.105 nucleotides) osmium peak negligible signal for intact DNA, remarkably increasing with number of breaks →more selective guanine peak (no Os,bipy treatment) considerable signal for intact DNA, slightly increasing DNA structural changes due to intercalation CQ conc.: a-0, b-10 mM, c-50 mM Due to intercalation (e.g., of chloroquine) in solution/during DNA immobilization DNA in the absence of intercalator: B-form, bases hidden in the double-helix interior, relatively far from the electrode surface DNA saturated with the intecalator (intDNA): untwisted and lengthened double helix, less deep grooves – bases closer to the surface, contacts between the surface and the base pair edges after removal of the intercalator, the intDNA conformation is preserved the adsorbed untwisted regions of intDNA yield the AV voltammetric peak 2