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Synthesis and delivery of novel fluorescently-labelled nucleotides and their nucleic acids for bio-analytical applications
Güixens Gallardo, Pedro ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Klán, Petr (referee)
1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...
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Bioapplications of novel nanostructured materials
Fučíková, Anna ; Valenta, Jan (advisor) ; Polívka, Tomáš (referee) ; Plášek, Jaromír (referee)
Title: Bioapplications of novel nanostructured materials Author: Anna Fučíková Department / Institute: Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Supervisor of the doctoral thesis: Doc. RNDr. Jan Valenta, Ph.D., Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Abstract: This work is aimed at development of new fluorescent labels based on silicon nanocrystals. Nanodiamonds and commercial CdSe quantum dots have been used as comparative materials. Silicon nanocrystals are relatively small (1-4 nm) compared to other studied nanomaterials. They are prepared by electro-chemical etching and their surface can be activated by various molecules which strongly influences luminescence properties. Luminescence quantum efficiency can be as high as 30 % and perfectly photostable even in biological environment. Si nanocrystals are biodegradable in a living organism within reasonable time scale and non-toxic. We are able to detect luminescence of single nanocrystals, even inside living cells, with use of our micro-spectroscopy apparatus. Nanodiamonds have weak luminescence; they are toxic at higher dosages and very stable in living bodies (without available technique how to remove them). Studied CdSe...
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Synthesis and delivery of novel fluorescently-labelled nucleotides and their nucleic acids for bio-analytical applications
Güixens Gallardo, Pedro ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Klán, Petr (referee)
1 Abstract The goals of the thesis were to synthesise novel fluorescently labelled nucleotides and the corresponding nucleic acids for bio-analytical applications as well as their delivery into cells. The thesis also aimed at the development of an effective method to inhibit non-templated incorporation of nucleotides. The problematic non-templated enzymatic incorporation of nucleotides is addressed by using several commercially available 5'-modified-oligonucleotides. The oligonucleotides (ONs) that we tested bore ortho twisted intercalating nucleic acid (oTINA), a trityl group, or biotin at the 5'-end. The modified ONs were used as templates in the enzymatic primer extension (PEX) experiments in the presence of either modified nucleotides or only natural deoxynucleoside triphosphates (dNTPs). The oTINA templates underwent PEX reaction using natural dNTPs and different DNA polymerases of the A or B family. In parallel, two types of fluorescent nucleoside derivatives were independently designed and synthesised. Firstly, we envisaged new fluorescent nucleotide tags containing the hexamethylated BODIPY moiety as a bright fluorescent label. Conversely, we focused on the improvement of fluorescent nucleotide probes sensitive to the viscosity or polarity. The fluorescently labelled methylated BODIPY nucleotides...
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Bioapplications of novel nanostructured materials
Fučíková, Anna ; Valenta, Jan (advisor) ; Polívka, Tomáš (referee) ; Plášek, Jaromír (referee)
Title: Bioapplications of novel nanostructured materials Author: Anna Fučíková Department / Institute: Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Supervisor of the doctoral thesis: Doc. RNDr. Jan Valenta, Ph.D., Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University in Prague Abstract: This work is aimed at development of new fluorescent labels based on silicon nanocrystals. Nanodiamonds and commercial CdSe quantum dots have been used as comparative materials. Silicon nanocrystals are relatively small (1-4 nm) compared to other studied nanomaterials. They are prepared by electro-chemical etching and their surface can be activated by various molecules which strongly influences luminescence properties. Luminescence quantum efficiency can be as high as 30 % and perfectly photostable even in biological environment. Si nanocrystals are biodegradable in a living organism within reasonable time scale and non-toxic. We are able to detect luminescence of single nanocrystals, even inside living cells, with use of our micro-spectroscopy apparatus. Nanodiamonds have weak luminescence; they are toxic at higher dosages and very stable in living bodies (without available technique how to remove them). Studied CdSe...
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