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Synthesis of functionalized nucleosides and nucleotides and their incorporation into DNA
Balintová, Jana ; Hocek, Michal (advisor) ; Stiborová, Marie (referee) ; Bartošík, Martin (referee)
This PhD thesis describes the synthesis of modified DNA containing electrochemical active labels such as anthraquinone, benzofurazane, azide and nitrophenyltriazole with the application in bioanalysis. The methodology of the construction of base-modified triphosphates based on Suzuki-Miyaura coupling of halogenated deoxynucleoside triphosphates with arylboronic acids, Sonogashira coupling of dNTPs with terminal acetylenes or triphosphorylation of modified nucleosides was developed. Enzymatic incorporation of functionalized dNTPs into DNA by primer extension experiment and the electrochemical properties of DNA bearing redox labels were studied by cyclic and square- wave voltammetry. At first anthraquinone was tested as an electrochemical label. Modified 2'-deoxynucleoside triphosphates bearing anthraquinone attached through an acetylene or propargylcarbamoyl linker at 5-position of 2'-deoxycytidine 5'-triphosphate and at the 7-position of 7-deaza-2'-deoxyadenosine-5'-triphosphate were prepared by Sonogashira cross-coupling. Enzymatic incorporation of the anthraquinone-labeled dNTPs into DNA by primer extension has been developed. The electrochemical properties of the anthraquinone- labeled nucleosides, dNTPs and DNA were studied by cyclic voltammetry which shows a reversible couple of peaks around...
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Searching for Electrochemical Reduction Mechanism of Azidophenyl DNA Labels
Daňhel, Aleš ; Trošanová, Zuzana ; Balintová, Jana ; Hocek, Michal ; Fojta, Miroslav
This contribution brings new information to sporadic scientific results concerned to electrochemical reduction of aromatic azides. Selected compounds used as perspective DNA labels or their structural constituents such as, 4-azidophenyltrifluoroboronic acid, 4-azidophenyl modified deoxycytidine and next probable analogous compounds and/or products of their reduction, were voltammetrically studied and compared to each other in order to reveal a mechanism of their electrochemical reduction at mercury electrodes in aqueous media. Preliminary results obtained by cyclic voltammetry at mercury and carbon based electrodes and by mass spectrometry of the products isolated from batch electrolysis on mercury pool by preparative chromatography are discussed.
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Magnetic Beads-Based Electrochemical Techniques for DNA-Protein Interaction Monitoring
Fojta, Miroslav ; Pivoňková, Hana ; Němcová, Kateřina ; Horáková Brázdilová, Petra ; Havran, Luděk ; Orság, Petr ; Vidláková, Pavlína ; Macíčková-Cahová, Hana ; Balintová, Jana ; Hocek, Michal
Electrochemical techniques, in connection with separation of nucleoprotein complexes at magnetic beads, are suitable for the monitoring of DNA-protein interactions. For the detection of complexes captured at the beads it is possible to utilize intrinsic electrochemical activity of the protein, intrinsic structure-selective signals of the DNA, or indicator DNA substrates tail-labeled with electroactive moieties.
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Electrochemical analysis of DNA using switchable redox moieties
Fojta, Miroslav ; Daňhel, Aleš ; Horáková Brázdilová, Petra ; Plucnara, Medard ; Pivoňková, Hana ; Havran, Luděk ; Vidláková, Pavlína ; Raindlová, Veronika ; Balintová, Jana ; Macíčková-Cahová, Hana ; Hocek, Michal
Labelling of DNA with electrochemically active moieties proved to be a convenient way to the development of electrochemical techniques for the sequence-specific DNA sensing. Through combinations of various labels differing in redox potentials, independent redox coding of different DNA sequences or individual nucleobases can be attained. Applications possibilities of electrochemistry in analysis of modified DNAs are further extended by facile monitoring of chemical conversion of reactive groups on DNA during post-labelling with ultimate redox labels. In addition, controlled in situ electrochemical conversions of specific intrinsic and extrinsic DNA components can be utilized to switch their electrochemical signals and improve signal resolution.
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Polymerase synthesis of base-modified DNA: New methods and new applications
Balintová, Jana ; Daďová, Jitka ; Kielkowski, Pavel ; Ménová, Petra ; Vaníková, Zuzana ; Riedl, Jan ; Raindlová, Veronika ; Fojta, Miroslav ; Hocek, Michal
Diverse base-modified oligonucleotides and double-stranded DNA molecules were prepared by polymerase incorporation of modified nucleoside triphosphates. The methods include primer extension, PCR, nicking enzyme amplification reaction and mixed incorporations. The modified nucleic acids were used in redox and fluorescence labelling and coding, as well as regulation of binding and cross-linking with proteins.
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