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Synthesis and studies of modified DNA: (i) development of DNA targeting molecular scissors and (ii) competitive enzymatic incorporation of base-modified nucleotides
Panattoni, Alessandro ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Fojta, Miroslav (referee)
In the first part of this work, a series of site-specific artificial metallonucleases (AMNs) was developed conjugating clamped-phenanthroline (Clip-Phen) copper complexes to triplex- forming oligonucleotides (TFOs). Several synthetic routes were explored for the synthesis of the TFO-AMNs hybrids, all sharing a copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction as the key step. As a consequence, building blocks for enzymatic or chemical synthesis of oligonucleotides (ONs) containing clickable groups, or already conjugated to the Clip-Phen ligand via CuAAC, were prepared. Two new alkynyl-linked nucleoside-5'-O-triphosphates (dNTPs) were designed and developed in order to obtain an efficient polymerase incorporation of clickable alkynyl-tethers into ONs and, at the same time, enhance the efficiency of CuAAC reactions on modified DNA. The relative 3'-O- phosphormaidites were also prepared in order to insert the same alkynyl-linkers into ONs via solid-phase synthesis. The AMN was linked at the 5'- or 3'-ends or in the middle of the TFO stretch, using diverse likers. The hybridization of all the synthesized TFOs with a target DNA duplex was studied. Finally, an extensive study of cleavage efficiency and specificity of the TFO-AMN conjugates towards the target DNA was performed, exploring the...
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Utilization of Catalytic Hydrogen Evolution in Electrochemical Analysis of Nucleic Acids
Fojta, Miroslav ; Daňhel, Aleš ; Špaček, Jan ; Havran, Luděk ; Šebest, Peter ; Orság, Petr ; Pivoňková, Hana ; Vosáhlová, J. ; Schwarzová-Pecková, K.
Catalysis of hydrogen evolution (CHE) at mercury in the presence of proteins was discoverd shortly after the introduction of polarography. In contrast, unmodified nucleic acids have not been reported to produce distinct signals due to the CHE to date. Chemically modified nucleic acids bearing certain extrinsic groups produce analytically useful signals due to hydrogen evloution catalyzed by the respective modifications. These species include (a) transition metal complexes, and (b) non-metal catalytically active organic moieties. In addition, the CHE has been reported to be invoved in guanine reduction process at the mercury-based electrodes.
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2D Condensation of 5-Fluorocytosine in Different Electrolytes
Fojt, Lukáš ; Fojta, Miroslav
Two-dimensional (2D) condensation and adsorption of 5-fluorocytosine on hanging mercury drop electrode in differnt electrolytes was investigated. We have used different sodium halide salts as basic electrolytes. The influence of electrolytes composition was displayed using differential capacitance measurements. Effects of ions present in used electolytes solution was remarkable. We observed huge change in 2D condensed layers in dependence on ions type.
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A Preliminary Study of Modification of 7-Deazaadenine with a Complex of Osmium Tetroxide with 2,2 '-Bipyridine
Vítová, Lada ; Havran, Luděk ; Fojta, Miroslav ; Šedo, O. ; Zdráhal, Z. ; Vespalec, Radim
Reactivity of a purine nucleic base analogue 7-deazaadenine (A*) in short oligodeoxynucleotide (ODN) towards an osmium tetroxide complex with 2,2'-bipyridine (Os, bipy) was investigated by means of electrochemistry, capillary electrophoresis and MALDI-TOF mass spectrometry. Electrochemical measurement, electrophoretic analyses and mass spectrometric analyses of reaction mixture proved that ODN with an A* residue yields an osmium adduct with properties similar to those exhibited by the well-known adduct of thymine.
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P36 VOLTAMETRIC DETECTION OF DNA DELETION ON PENCIL ELECTRODE
Haroniková, Lucia ; Špaček, Jan ; Fojta, Miroslav
In this work, we present a new qualitative approach of detection of PCR products using electrochemistry on pencil electrodes. PCR products with an incorporated biotin-labeled dNTP (dCTP in this study) are detected via conjugated streptavidinealkaline phospatase. 1-Naphthyl phosphate (1-NP), which is dephosphorylated by alkaline phosphatase to release 1-naphthol, was used as a substrate in electrochemical detection of the PCR product. Voltammetric measurement on disposable pencil electrode is a very cheap and easy to use method. The system was optimized for plasmid DNA PCR product with potential to detect human genome DNA products and possible application in gene deletion monitoring.
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Study on the Electrophoretic Behaviour of Short Oligodeoxyribonucleotides in Fused Silica Capillaries. A Preliminary Communication
Vítová, Lada ; Fojta, Miroslav ; Vespalec, Radim
Effects of background electrolyte pH, composition and concentration on the electrophoretic transport of short oligodeoxyribonucleotides (ODNs) in fused silica capillaries have been investigated in the presented study. Mobilities and separation efficiencies of ODNs peaks varied with all three background electrolyte characteristics. Just changes in the background electrolyte characteristics and acid-base properties of ODNs are not sufficient for a systematic explanation of the observed variations. Despite the indicated complexity of ODNs electrophoretic behaviour, we show that very high separation efficiencies of ODNs peaks can be achieved in background electrolytes of physiological pH.
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