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Pavlína Vidláková MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA STUDIUM ELEKTROCHEMICKÝCH VLASTNOSTÍ SYNTETICKÝCH OLIGONUKLEOTIDŮ A DNA MODIFIKOVANÝCH REDOX-AKTIVNÍMI SKUPINAMI Disertační práce Pavlína Vidláková Vedoucí práce: Doc. RNDr. Miroslav Fojta CSc. Brno 2015 Bibliografický záznam Autor: Mgr. Pavlína Vidláková Studium elektrochemických vlastností syntetických Název práce: oligonukleotidů a DNA modifikovaných redox- aktivními skupinami Studijní program: Fyzika Studijní obor: Biofyzika Vedoucí práce: Doc. RNDr. Miroslav Fojta CSc. Akademický rok: 2014/15 Počet stran: 53+57 Klíčová slova: DNA, elektrochemie, elektrochemické značení, biosensor Bibliographic Entry Author Mgr. Pavlína Vidláková Study of electrochemical properties of of synthetic Title of Thesis: oligonucleotides and DNA modified with redox-active moieties Degree programme: Physics Field of Study: Biophysics Supervisor: Doc. RNDr. Miroslav Fojta CSc. Academic Year: 2014/15 Number of Pages: 53+57 Keywords: DNA, electrochemistry, electrochemical labeling, biosensor Poděkování Na tomto místě bych chtěla poděkovat svému školiteli Doc. RNDr. Miroslavu Fojtovi CSc. a také Mgr. Luďkovi Havranovi Dr. za jejich odborné vedení a cenné rady a také za připomínky při zpracování této práce. Déle děkuji kolegům z Biofyzikálního ústavu za příjemné pracovní prostředí a veškerou pomoc, které se mi od nich dostalo. Prohlášení Prohlašuji, že jsem svoji rigorózní práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány. Brno 6. března 2015 ……………………………… Pavlína Vidláková © Pavlína Vidláková, Masarykova Universita, 2015 Abstrakt V této disertační práci se věnujeme elektrochemické analýze nukleotidových sekvencí s využitím elektrochemických značek a vlastnostem těchto značek. Teoretická část práce je zaměřena na shrnutí poznatků týkajících se elektrochemického chování nukleových kyselin a oligonukleotidů s využitím různých typů elektrod a elektrochemických metod. Dále jsou zde popsány metody značení nukleových kyselin elektroaktivními značkami a také příprava a použití DNA-biosenzorů. Experimentální část je přiložena ve formě pěti publikací otištěných v impaktovaných časopisech, viz přílohy 1-5. V kapitole Výsledky a diskuze jsou shrnuty a stručně komentovány výsledky obsažené v těchto článcích. Tyto články jsou zaměřené především na přípravu nových elektrochemickým značek (antrachinonu, nitrofenylu, benzofurazanu a butylakrylátu) využitelných pro značení DNA a přípravu biosenzorů a detailní studium elektrochemického chování těchto látek a to jak samostatně, tak v různých kombinacích. Tyto značky byly pomocí metody prodlužování primeru inkorporovány do molekul oligonukleotidů a takto připravené oligonukleotidy byly opět elektrochemicky studovány. Poslední článek je zaměřen na elektrochemické studium DNA modifikované kancerostatikem cisplatinou. Cisplatina se kovalentně váže na DNA, nejčastějším vazebným místem jsou dinukleotidové motivy GG a AG. Při elektrochemické redukci komplexních sloučenin platiny na rtuťových elektrodách dochází ke katalytickému vylučování vodíku. Tento proces je možné využít pro citlivé stanovení modifikované DNA. Abstract In this thesis we deal with electrochemical analysis of nucleotide sequences using electroactive labels and properties of these labels. The theoretical part is concentrated on summary of findings regarding of electrochemical behavior of nucleic acid and oligonucleotides using of various types of electrodes and electrochemical methods. Further are there methods of DNA labeling with using electroactive tags and methods of preparation of DNA-biosensors. Experimental part of the thesis encompasses five papers published in international peer- reviewed journals (see appendices 1-5). The results included in these papers are summarized and briefly commented in the chapter Results and Discussion. These papers are concentrated on preparation of new electroactive labels for DNA and for preparation of DNA-biosensors, and on studying electrochemical behavior of these labels separately or in various combinations of several types of labels simultaneously. These tags were incorporated into oligonucleotides with using primer extension and these oligonucleotides were electrochemically studied. The last paper deals about electrochemical analysis of cisplatin modified DNA. Cisplatin binds covalently on DNA (especially on dinucleotide motifs GG and AG). Electrochemical reduction of cisplatin-DNA adduct at mercury electrodes is accompanied by catalytic hydrogen evolution. This process can be used for sensitive analyses of modified DNA. Obsah Seznam zkratek………………………………………………………………………….…..…8 Úvod………………………………………………………………………………………..…10 Literární přehled……………………………………………………………………….….…..12 1. Elektrochemické metody…………………………………………………….…....12 1.1 Pracovní elektrody………………………………………………….….…12 1.1.1 Rtuťové pracovní elektrody…………………………….….….12 1.1.2 Pevné pracovní elektrody……………………………….….....14 1.2 Referentní a pomocné elektrody…………………………………….…....15 1.3 Elektroanalytické metody…………………………………………..….…16 1.4 Rozpouštěcí a přenosové techniky……………………………….……....19 2. Elektrochemické vlastnosti nukleových kyselin………………………….………21 2.1 Elektrochemické chování DNA na rtuťových elektrodách………………21 2.1.1 Redoxní děje na rtuťových elektrodách……...…………....….21 2.1.2 Adsorpčně-desorpční děje na rtuťových elektrodách...…….…22 2.2 Elektrochemické chování DNA na uhlíkových elektrodách……………..24 3. Elektrochemické značení nukleových kyselin……………………………………25 3.1 Nekovalentě se vázající redoxní indikátory……………………………...26 3.2 Kovalentně se vázající elektrochemické značky…………………………26 3.3 Enzymatická inkorporace elektrochemicky značených nukleotidů……...28 3.4 Magnetoseparační techniky………………………………………………30 4. Elektrochemické biosenzory……………………………………………………...31 4.1 Elektrochemické biosenzory pro detekci hybridizace DNA……………..31 4.1.1 Imobilizace hybridizační sondy na povrchu elektrod………....32 4.1.2 Detekční principy využívané v elektrochemických senzorech pro hybridizaci DNA………………………………………….33 4.2 Elektrochemické senzory pro detekci mutací a polymorfismů…………..34 Cíle disertační práce…………………………………………………………………………..36 Seznam publikací……………………………………………………………………… ……37 Výsledky a diskuse……………………………………………………………………………38 Závěr………………………………………………………………………………………….49 Seznam literatury……………………………………………………………………………..50 Přílohy………………………………………………………………………………………...54 7 Seznam zkratek A, C, G, T – adenin, cytosin, guanin, thymin ACV - voltametrie s vkládaným střídavým napětím AdSV – adsorptivní rozpouštěcí voltametrie AdTSV – adsorptivní přenosová rozpouštěcí voltametrie Aox, Gox – oxidační píky adeninu a guaninu ASV – anodická rozpouštěcí voltametrie CPSA - chronopotenciometrie s konstantním proudem CSV – katodická rozpouštěcí voltametrie CV – cyklická voltametrie DNA (ds, ss, sc) – deoxyribonukleová kyselina (dvouřetězcová, jednořetězcová, superhelikální) dNTP – deoxyribonukleosid trifosfát DPP – diferenční pulsní polarografie DPV – diferenční pulsní voltametrie HMDE – visící rtuťová kapková elektroda LSV - voltametrie s lineárně se měnícím potenciálem MeSAE – pevná amalgámová elektroda (z amalgámu kovu Me) NK – nukleová kyselina ON – oligonukleotid Os,L – komplexy oxidu osmičelého PEX – prodlužování primeru (primer extension) 8 PGE – elektroda z pyrolytického grafitu RNA – ribonukleová kyselina SAM – samoorganizovaná monovrstva (self-assambled monolayer) SWV - voltametrie se superponovaným pravoúhlým napětím TdT – terminální deoxynuxleotidyl transferáza 9 Úvod Počátek elektrochemie nukleových kyselin spadá do druhé poloviny padesátých let dvacátého století. V roce 1958 uveřejnil E. Paleček práci, ve které pomocí oscilografické polarografie se střídavým proudem prokázal elektroaktivitu DNA (1). V roce 1961 byla publikována první práce, která se zabývala adsorpcí DNA na povrch rtuťové elektrody (2). Výsledky impedančního měření ukázaly, že je DNA povrchově aktivní látkou schopnou adsorpce na povrchu elektrody a při vkládání negativních potenciálů podléhá charakteristickým adsorpčně/desorpčním dějům. Tyto objevy vedly k dalšímu zkoumání elektrochemické a povrchové aktivity nukleových kyselin. Ukázalo se, že elektrochemické metody mohou přinášet informace jak o přítomnosti a koncentraci nukleových kyselin, tak i o jejich sekundární a terciální struktuře (3-7). Zpočátku byl vývoj v oblasti elektrochemie nukleových kyselin limitován jak dostupnou instrumentací, tak i omezenými možnostmi přípravy vhodného experimentálního materiálu. Tato situace se výrazně změnila až v posledním desetiletí 20. století, kdy došlo k velkému pokroku jak v biologických i medicínských vědách, tak i k rozvoji analytických zařízení i metod. V současné době se využívá široké spektrum elektrochemických metod, které umožňují stanovení stopových množství nukleových kyselin ve velmi malých objemech vzorků. Ke snížení spotřeby analyzovaného materiálu přispělo mimo jiné zavedení adsorptivních rozpouštěcích technik ve spojení se rtuťovými nebo uhlíkovými elektrodami. Tyto techniky spočívají v akumulaci vzorku na elektrodu před samotným měřením, čím je dosaženo větší citlivosti stanovení. Adsorpce nukleových kyselin na elektrodu je možné provést z velmi malého (jednotky mikrolitrů) množství vzorku, což snižuje spotřebu analytického materiálu (8). Přestože přirozená DNA je elektrochemicky aktivní, je pro některé analytické aplikace vhodné využít elektrochemického značení. To obvykle dovoluje citlivější detekci, než jaká by byla dosažena měřením vlastních signálů DNA, poskytuje signály v přístupnějších potenciálech mimo oblast vybíjení
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