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Approaches to the enzymatic synthesis of hypermodified DNA polymers
Ondruš, Marek
The aim of this thesis was to synthesize new series of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing hydrophobic modifications, use them in enzymatic synthesis of fully-modified DNA and study its bio-physical properties. In the first part of the thesis, a set of four 2'-deoxynucleosides bearing a linear or branched alkane, indole or phenyl group were synthesized by Sonogashira cross-coupling reactions using alkynes and iodinated nucleosides. Ethynyl linkers of corresponding nucleosides were reduced by catalytic hydrogenation to obtain another four nucleosides bearing the modifications through alkyl linker. All eight nucleosides were then transformed into 2'-deoxynucleoside triphosphates (dNTPs) by Yoshikawa phosphorylation and individually tested as substrates for polymerase synthesis by primer extension (PEX). Their combinations were systematically tested to generate DNA containing one or even four modified nucleotides. It was possible to replace all four canonical nucleotides with hydrophobically-modified counterparts and thus synthesize DNA with high density of modifications. Nevertheless, only nucleotides bearing modifications through rigid ethynyl linker were suitable for synthesis of longer hypermodified DNA. Nucleotides with more flexible alkyl linker destabilized duplex during...
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Aptamers application in research and diagnostics
Neumanová, Alice ; Svobodová, Zuzana (advisor) ; Rathouská, Jana (referee)
The bachelor thesis deals with affinity molecules that are able to specifically bind to the target molecule. The first chapter is devoted to antibodies, i.e., affinity molecules of protein nature. It discusses their structure, development, production, and use. The main focus of the thesis is on aptamers, or short stretches of single-stranded DNA or RNA, which are often referred to as synthetic alternatives to antibodies. They have similar functions to antibodies but differ in structure, development, and production. Because of their similarity to antibodies, aptamers are used in various detection technologies to diagnose diseases. Aptamers are still in the research phase, but the first commercially available aptamer diagnostic kits are slowly starting to appear on the market. The final chapter compares the pros and cons of the two biggest rivals in the field of affinity molecules - antibodies and aptamers. Key words: aptamers, antibodies, nanoantibodies, affimers, diagnostics, research
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Approaches to the enzymatic synthesis of hypermodified DNA polymers
Ondruš, Marek ; Hocek, Michal (advisor) ; Janeba, Zlatko (referee) ; Zimčík, Petr (referee)
The aim of this thesis was to synthesize new series of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing hydrophobic modifications, use them in enzymatic synthesis of fully-modified DNA and study its bio-physical properties. In the first part of the thesis, a set of four 2'-deoxynucleosides bearing a linear or branched alkane, indole or phenyl group were synthesized by Sonogashira cross-coupling reactions using alkynes and iodinated nucleosides. Ethynyl linkers of corresponding nucleosides were reduced by catalytic hydrogenation to obtain another four nucleosides bearing the modifications through alkyl linker. All eight nucleosides were then transformed into 2'-deoxynucleoside triphosphates (dNTPs) by Yoshikawa phosphorylation and individually tested as substrates for polymerase synthesis by primer extension (PEX). Their combinations were systematically tested to generate DNA containing one or even four modified nucleotides. It was possible to replace all four canonical nucleotides with hydrophobically-modified counterparts and thus synthesize DNA with high density of modifications. Nevertheless, only nucleotides bearing modifications through rigid ethynyl linker were suitable for synthesis of longer hypermodified DNA. Nucleotides with more flexible alkyl linker destabilized duplex during...
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In Vitro Selection of Aptamers for Methionine Sulfoxide
Jureček, Matěj ; Míšek, Jiří (advisor) ; Bařinka, Cyril (referee)
Oxidation of methionine to methionine sulfoxide in proteins is considered one of important post-translational modifications of proteins. This modification can activate and also inhibit functions of many proteins and it is a part of regulation mechanisms of various (patho)physiological processes. For further research of the effects of methionine oxidation in proteins it would be very helpful to find its bioindicator. So far however, there has not been found any such antibody, nor any of its alternatives. This thesis was concerned with the search of ssDNA aptamer specific for methionine sulfoxide by the method of in vitro selection (SELEX). Several conditions for in vitro selection of methionine sulfoxide were tested in this diploma thesis. None of them led to the enrichment of the starting oligonucleotide pool and no selective aptamer for methionine sulfoxide has been found. Such results don't necessarily point to the impossibility of finding such aptamer, but the conventional methods used in this thesis weren't suitable for this task. In a control in vitro selection there has been found an enriched ssDNA pool for sulforhodamine B as a ligand. Sequencing of clones of this enriched pool has shown oligonucleotides with G-rich sequences, which is typical for already published aptamers for sulforhodamine B.
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