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Modified ribonucleotides as building blocks for enzymatic construction of functionalized RNA or as antiviral compounds
Milisavljević, Nemanja ; Hocek, Michal (advisor) ; Baszczyňski, Ondřej (referee) ; Krečmerová, Marcela (referee)
The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...
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Enzymatic synthesis of DNA modified in the minor groove
Matyašovský, Ján ; Hocek, Michal (advisor) ; Hlaváč, Jan (referee) ; Urban, Milan (referee)
In the first part of the thesis, a series of six modified 2'-deoxyadenosine triphosphates, bearing small functional groups (chloro, amino, methyl, vinyl, ethynyl and phenyl) at position 2 of adenine, was designed and synthesised. They were then tested as substrates for DNA polymerases in enzymatic synthesis of minor-groove modified DNA. The 2-phenyl modified dATP was the only triphosphate unable to be incorporated, meaning that the phenyl group is already too big for minor-groove incorporations. All of the other tested nucleotides were good substrates for tested DNA polymerases [KOD XL, Vent(exo-) and Bst LF] affording minor- groove modified DNA bearing one or four modifications. The vinyl- and ethynyl-modified DNAs were then used for post-synthetic modification of DNA minor groove with fluorescent labels utilising click reactions. Ethynyl group reacted in copper-catalysed alkyne-azide cycloaddition (CuAAC), whereas the vinyl group participated in thiol-ene reaction. This procedure allowed for the attachment of big functional groups otherwise unable to be installed into the DNA minor groove using direct enzymatic incorporation. The second part of the thesis was devoted to the study of 2-alkylamino-2'- deoxyadenosine triphosphates and their use in enzymatic synthesis of base-modified ONs and DNA....
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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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Construction of modified DNAs with selected reactive or protective groups
Vaníková, Zuzana ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Zimčík, Petr (referee)
This PhD thesis is focused on the synthesis of DNA modified with photocleavable 2- nitrobenzyl protecting groups in major groove and its applications in the regulation of gene expression in the level of transcription. In the first part of my thesis, the synthesis of photocaged 2'-deoxyribonucleosides triphosphates and their photolysis to unprotected 5-hydroxymethylated nucleotides is described. All prepared nucleoside triphosphates were good substrates for their enzymatic incorporation into DNA. Synthesized 5-(2-nitrobenzyloxy)methyl-2'-deoxyuridine-5'- monophosphate (dUNBMP) and DNA with one 5-(2-nitrobenzyloxy)methyl- modification in the sequence were used for the detailed kinetic studies of photocleavage reactions. In the second part of the thesis, the series of modified DNAs with specific sequences were prepared by primer extension (PEX) and/or polymerase chain reaction (PCR). A cleavage of prepared modified DNAs was studied by selected restriction endonucleases (REs). In all cases, the nitrobenzylated DNA fully resist the cleavage by REs. The deprotection/ photocleavage conditions for nitrobenzylated DNA were studied in the case of DNAs with positive restriction endonuclease digestion of hydroxymethylated DNA. The resulting photocleaved DNA was fully digested by REs, therefore 2-nitrobenzyl...
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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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New redox labels for DNA
Simonova, Anna ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Vyskočil, Vlastimil (referee)
The aim of my thesis was the synthesis of the modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing electrochemically oxidizable labels and their incorporation into DNA for the application in bioanalysis. In the first part of my thesis, I developed the synthesis of modified dNTPs bearing 2,3- dihydrobenzofuran (DHB) or 2-methoxyphenol (MOP) labels at 5-position of 2'- deoxycytidine 5'-O-triphosphate and at the 7-position of 7-deaza-2'-deoxyadenosine 5'-O- triphosphate by Suzuki-Miyaura cross-coupling reactions. Then modified dNTPs were used as substrates for DNA polymerases in enzymatic synthesis of modified DNA by PCR and primer extension. Electrochemical properties of the DHB and MOP-labeled nucleosides, dNTPs and DNA were studied by using of a square-wave voltammetry (SWV) at the pyrolytic graphite electrode (PGE) giving signals of MOP oxidation around 0.5 V and DHB oxidation around 0.85 V. The use of DHB group in combination with other electrochemical active labels was limited by close position of its oxidation peak to the signals of oxidation of natural nucleobases, whereas MOP moiety was successfully used for redox coding of nucleobases in combination with aminophenyl or benzofurazane label giving two independently readable redox signals in each case. In the second part of this...
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Preparation and enzymatic incorporation of deoxyribonucleic triphosphates derived from pyrimido[4,5-b]indol to DNA using selected polymerases.
Bosáková, Andrea ; Konvalinka, Jan (advisor) ; Hodek, Petr (referee)
This bachelor thesis deals with the synthesis of pyrimido[4,5-b]indole 2'- deoxyribonucleosides and their following trifosforylation. Overall there three new analogues 2'-deoxyadenosine triphosphate with benzen ring were prepared. Furthermore, the ability of DNA polymerase to incorporate in total four modified 2'-deoxyribonucleosides into the oligonucleotide strand by primer extension (PEX) method was observed. All the modified 2'-deoxyribonucleotides were incorporated into the oligonucleotide strand, however the success of the subsequent elongation was different accoring to the DNA polymerase that was used and according to the substitution in position 6 in the structure of substrate. Key words nucleosides, modified nucleotides, DNA polymerase, PEX reaction
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Determination of adenosine triphosphate and adenosine diphosphate in real samples
Černá, Martina ; Coufal, Pavel (advisor) ; Zima, Jiří (referee)
The aim of the diploma thesis was to find optimal conditions of high pressure liquid chromatography for the detection and quantification of two common nucleotides, namely adenosine diphosphate and adenosine triphosphate, as well as to perform an analysis of these in real life samples of citrus fruits and plant extracts. Further aim of the project was to determine the limits of detection and quantification of adenosine diphosphate and adenosine triphosphate under the optimized conditions and using these to compare the sensitivity of given detectors. To achieve this HPLC-UV, capillary HPLC-DAD and HPLC-MS apparatus were used. With the help of HPLC with UV detection and capillary HPLC with diode array detector, the calibration curves of the mixture of analytes were measured and the limits of detection as well as quantification of adenosine diphosphate and adenosine triphosphate were determined. Separation of the analytes up to the base line using HPLC-UV and capillary HPLC-DAD was achieved under the conditions of ion pairing chromatography. Column C18 was chosen as an appropriate column. The mobile phase included phosphate buffer, acetonitrile and tetrabutylammonium bisulphate as an ion pairing reagent. The separation was performed with gradient elution. Conditions for analysis using LC-MS were...
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Analysis of adenosine triphosphate and adenosine diphosphate by HPLC-MS/MS
Černá, Martina ; Coufal, Pavel (advisor) ; Bosáková, Zuzana (referee)
In this bachelor thesis, ADP and ATP samples were analysed and detected with HPLC- MS/MS method. Approximate limit of detection (LOD) for these particular substances were found and their values were compared with the LOD values published in the literature obtained via the same methods and under very similar experimental conditions. Our limits of detection for nucleotides were comparable with the limits described in the literature. Mass spectrometry analysis was performed in the positive and the negative mode of multiple reaction monitoring analysis and electrospray was used for the analyte ionization. The optimal conditions for high performance liquid chromatography of ATP and ADP analysis were acquired on a ZIC - HILIC column with the mobile phase of 75:25 (v/v) acetonitrile / 10 mM ammonium acetate. Ammonium acetate buffer was adjusted to pH of 7.15 and the separation was done under the isocratic elution.
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