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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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Functional analysis of posttranscriptional gene regulation by TENT5A in biomineralization and metabolism
Aranaz Novaliches, Goretti ; Sedláček, Radislav (advisor) ; Hovořáková, Mária (referee) ; Tencerová, Michaela (referee)
Non-canonical poly-A polymerases, such as TENT5A, belong to the Terminal nucleotidyl transferases (TENTs) family and are crucial for mRNA protection, stability, and translation. A Tent5a knock-out (KO) mouse model was generated in our laboratory, which exhibited a phenotype in teeth, skeleton structure, and metabolism. In my PhD project, I aimed to characterize the molecular mechanism underlying these phenotypes and explore their potential connection to rare human diseases. I focused on the biological function of Tent5a gene in enamel development (amelogenesis) and mRNA stabilization. Micro-computed tomography and scanning electron microscopy revealed that Tent5a KO mice displayed thin, hypomineralized enamel with disrupted microstructure, a condition known as Amelogenesis imperfecta. Direct mRNA sequencing demonstrated that TENT5A is responsible for polyadenylation of amelogenin (AmelX) and other secreted proteins, leading to a shortened poly-A tail in Tent5a KO ameloblasts. Moreover, Tent5a KO mice disclosed impaired self- assembly of enamel matrix proteins (EMPs) such as AMELX and ameloblastin (AMBN), leading to compromised hydroxyapatite deposition and enamel formation. In addition to its role in teeth, I investigated the physiological functions of EMPs in other tissues, considering that EMP...
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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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Modification of nucleic acids by reactive groups for bioconjugations and cross-linking with lysine-containing peptides and proteins
Ivancová, Ivana
In the first part of this thesis, I developed reactive DNA probe for selective cross-linking with lysine residues of DNA-binding proteins. I synthesized 2'-deoxycytidine 5'-O-mono- and triphosphate bearing squaramate moiety tethered to the position 5 via propargylamine linker. The monophosphate was used as a model compound to test the reactivity of this mixed squaramate in cross-linking reactions with lysine and short lysine containing peptides. Squaramate modified 2'-deoxycytidine 5'-O-triphosphate was found to be suitable substrate for KOD XL polymerase in both PEX and PCR synthesis of modified DNA. Squaramate modified DNA forms stable diamide linkage with primary amines. I tested the reactivity of this DNA probe in bioconjugation reactions with sulfo-Cy5-amine and lysine containing peptides. Afterwards, squaramate-linked DNA was successfully cross-linked with lysine rich histone proteins. This reactive squaramate modified nucleotide showed potential for following bioconjugation reactions of nucleic acids with amines or lysine containing peptides and proteins without the need of external reagent. Based on positive results of experiments with squaramate modified DNA, in the second part of the thesis I developed and synthesized squaramate modified ribonucleotide to study cross- linking with RNA...
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Modification of nucleic acids by reactive groups for bioconjugations and cross-linking with lysine-containing peptides and proteins
Ivancová, Ivana ; Hocek, Michal (advisor) ; Míšek, Jiří (referee) ; Urban, Milan (referee)
In the first part of this thesis, I developed reactive DNA probe for selective cross-linking with lysine residues of DNA-binding proteins. I synthesized 2'-deoxycytidine 5'-O-mono- and triphosphate bearing squaramate moiety tethered to the position 5 via propargylamine linker. The monophosphate was used as a model compound to test the reactivity of this mixed squaramate in cross-linking reactions with lysine and short lysine containing peptides. Squaramate modified 2'-deoxycytidine 5'-O-triphosphate was found to be suitable substrate for KOD XL polymerase in both PEX and PCR synthesis of modified DNA. Squaramate modified DNA forms stable diamide linkage with primary amines. I tested the reactivity of this DNA probe in bioconjugation reactions with sulfo-Cy5-amine and lysine containing peptides. Afterwards, squaramate-linked DNA was successfully cross-linked with lysine rich histone proteins. This reactive squaramate modified nucleotide showed potential for following bioconjugation reactions of nucleic acids with amines or lysine containing peptides and proteins without the need of external reagent. Based on positive results of experiments with squaramate modified DNA, in the second part of the thesis I developed and synthesized squaramate modified ribonucleotide to study cross- linking with RNA...
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Non-coding RNAs in oocyte and early embryo
Aleshkina, Daria ; Šušor, Andrej (advisor) ; Staněk, David (referee) ; Krylov, Vladimír (referee)
Once considered as 'transcriptional noise' noncoding RNAs (ncRNAs) nowadays are known to be key molecules in major cellular processes. NcRNAs are expressed at very high levels as only 2% of transcribed genome corresponds to protein-coding RNAs in higher eukaryotes. Various ncRNAs are known to have structural, functional, or regulatory roles, but the influence of the majority of non-coding transcripts is still unclear. Among ncRNAs, long ncRNAs (lncRNAs, longer than 200 bp) are of particular interest. LncRNAs do not have a uniform function but many studies observed lncRNA-based regulations at the transcriptional and translational levels. Therefore, novel lncRNAs could specifically fine-tune protein synthesis in the highly differentiated cell types. Particularly, fully-grown mammalian oocyte and early embryo require precisely controlled translation of maternal transcripts to coordinate meiotic progression and early embryo development while transcription is silent. We aimed to study the involvement of ncRNAs in protein synthesis and consequent influence on the oocyte and early embryo physiology. For the first time, we analysed the expression and distribution of several ncRNAs, namely Brain cytoplasmic RNA 1 (BC1), lncRNA in Oocyte Specifically Expressed (Rose), RNA Component of 7SK Nuclear...
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RNA secondary structure conservation identification module for rPredictor
Pešek, Jan ; Hoksza, David (advisor) ; Škoda, Petr (referee)
The issue of comparing ribosomal RNA secondary structures is currently an open research problem. Goal of this work is to design and implement program comparing so called mutual conservancy of a group of rRNA secondary structures. This program is included as a new module of an existing system called rPredictor consisting of the structure database and an algorithm that predicts these structures. This thesis covers an introduction into the problem of secondary structures conservancy identification and summarizes known approaches towards the problem. The result of this work is a stand alone program for the secondary structure comparison and also a new analytic module of the rPredictor system, where the secondary structure comparison program can be used for comparison of the structures from the rPredictor database. Powered by TCPDF (www.tcpdf.org)
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Aptamers affecting eIF4F subunits
Kopperová, Dana ; Feketová, Zuzana (advisor) ; Schierová, Michaela (referee)
Eukaryotic translation initiation factor 4F comprises of three subunits - eIF4A, eIF4G and eIF4E. These subunits play a key role in translation initiation. On its surface eIF4G binds other translation factors. eIF4A is RNA dependent helicase that is crucial for translation initiation. Affinity of eIF4E towards cap is enhanced if it is in complex with eIF4G. Specific molecules with high affinity were prepared by Selex. These molecules that bind various surfaces and different molecules in the cell are called aptamers. Aptamer binding eIF4A inhibits ATP hydrolysis. Activity of eIF4G may be affected by more aptamers but all resulting in translation inhibition. Aptamer binding eIF4E also inhibits translation by hindering cap binding. All of these translation factors may be detected in non-standard values in tumors. Aptamers against these proteins might bring a solution in the tumor treatment.
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Analysis of selected secondary structures of nucleic acids
Skružný, Petr ; Mokrejš, Martin (advisor) ; Drda Morávková, Alena (referee)
This work introduces a database of experimentally verified structures of nucleic acids which were collected from published scientific literature. The database is annotated and the structures are analysed from the perspective of quality and it was found that the experimentally obtained data are not always sufficient - their supporting evidence is often limited and their quality is not convincing. This work also discusses some of the problems, that can be encountered when the structures are experimentally probed. Contents of the database were compared to the RFAM database and despite of its small range it contains 80 new structures. The complete database of 166 structures can be possibly used to optimise software used to predict derived structures of nucleic acids. Furthermore, the work presents several possible ways of improvement of the quality of contained structures.
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