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Reactive modifications of RNA for bioconjugations with proteins and new enzymatic methods for the synthesis of base-modified RNA
Brunderová, Mária ; Hocek, Michal (advisor) ; Míšek, Jiří (referee) ; Vaňáčová, Štěpánka (referee)
This doctoral thesis focuses on the enzymatic synthesis of base-modified RNA probes with diverse functional groups, including reactive cross-linking, hydrophobic and fluorescent moieties, or affinity tags. The construction of nucleobase-modified oligonucleotides is accomplished either through conventional in vitro transcription with T7 RNA polymerase or by an innovative approach leveraging engineered mutant DNA polymerases and primer extension reaction (PEX). In the first section of the thesis, a novel ribonucleoside triphosphate building block with reactive chloroacetamide functionality was synthesised using an aqueous Pd-catalysed Sonogashira cross- coupling reaction, directly applied on iodinated nucleotide. The chloroacetamide modified triphosphate was then tested as a putative substrate for T7 RNA polymerase in in vitro transcription reaction, aiming to construct RNA probes with one or multiple reactive groups. The selectivity of chloroacetamide- modified RNA for thiol-, or cysteine-, and histidine-containing (bio)molecules was demonstrated by model bioconjugation reactions and cross-linking experiments with three RNA-binding proteins of diverse structures and functions. The efficient formation of RNA-protein covalent adducts was confirmed by western blot or gel, and mass spectrometry analyses...
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Modified nucleotides and DNA for electrochemical labelling and defined display of small molecules
Krömer, Matouš ; Hocek, Michal (advisor) ; Křen, Vladimír (referee) ; Vrábel, Milan (referee)
This thesis is focused on enzymatic construction of DNA probes for electrochemical labelling, bioconjugations and, in the final part, building on knowledge gathered in previous chapters, it describes a method useful for construction of highly functionalized base-modified DNA enabling defined multivalent display of glycosides. In first chapter, a chemical route to diol-bearing nucleotides was found. Sonogashira reaction facilitated access to alkyne-tethered diols and subsequent catalytic hydrogenation, described for the first time in the literature, provided protection-free method for obtaining nucleotide diols tethered via flexible sp3 hybridized linker. Cleavage of alkane-linked, but not alkyne-linked, nucleotide diols yielded aliphatic nucleotide aldehyde. All nucleotides were found to be good substrates for KOD XL DNA polymerase in both primer extension and polymerase chain reaction, apart from aldehyde-linked dUCHO TP nucleotide, which performed well in PEX reaction, but gave PCR products only in a mixture with natural dTTP. This could be overcome by cleavage of diol-modified DNA, which also yielded aldehyde-functionalized dsDNA. All reactive probes were examined for bioconjugations with fluorescent hydrazine, reductive amination with lysine or lysine-containing peptides or other molecules...
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Novel fluorescent nucleotides for metabolic labelling and for the construction of DNA probes
Kuba, Miroslav
The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified...
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Bioorthogonal reactions on DNA for regulation of transcription
Chakrapani, Aswathi ; Hocek, Michal (advisor) ; Zimčík, Petr (referee) ; Vrábel, Milan (referee)
This PhD thesis describes the design and synthesis of photocaged or glucosylated derivatives of epigenetic 5-(hydroxymethyl)pyrimidine-modified nucleotides and DNA using chemical and enzymatic methods and the studies on their regulation of gene expression in bacterial (Escherichia coli RNA polymerase) in vitro transcription level. In the first part of the thesis, the design and syntheses of 5-(nitrobenzyloxymethyl)-2'-deoxyuridine (dUNB) and -cytidine (dCNB) phosphoramidites are described. These photocaged nucleoside phosphoramidite building blocks were used in the automated solid-phase synthesis of oligonucleotides (ONs) modified at specific positions. The ONs were used as forward primers in a polymerase chain reaction (PCR) to construct DNA templates modified at specific sites of the promoter region. The specific site photocaged DNA was then irradiated with light to result in the corresponding specific site 5-(hydroxymethyl)-modified DNA. Bacterial in vitro transcription studies of both the specific site photocaged and uncaged DNA were carried out. The incorporation of the photocaged epigenetic pyrimidine nucleotides at the -35 region of the promoter region of the template DNA inhibited transcription partially while the presence of the same outside the -35 region did not have any significant...
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Modifications of DNA by reactive groups for bioconjugations and cross-links with arginine-containing peptides and proteins
Leone, Denise Liu ; Hocek, Michal (advisor) ; Urban, Milan (referee) ; Vrábel, Milan (referee)
This PhD thesis describes the development and the synthesis of DNA-reactive probes bearing 1,3- diketone or phenylglyoxal moieties which can be used for cross-linking with arginine-containing peptides or proteins. The general strategy was based on the functionalization of the position 5 of pyrimidines via aqueous Sonogashira coupling or click reaction (CuAAC) with reactive building blocks to synthesize modified 2'-deoxyribonucleoside monophosphates and triphosphates (dNMPs/dNTPs). The following step was the enzymatic incorporation of such functionalized dNTPs into DNA via primer extension (PEX). In parallel, the monophosphate derivatives were used as model compounds for reactions with arginine and arginine-containing peptides to test the reactivity of the probes. For the reactive candidates, model reactions on the modified-DNA with arginine or Arg-containing peptides were performed and the DNA-probes were used in cross- linking reactions with Arg-rich DNA-binding proteins. The development of such probes was not straightforward but required the investigation of diverse candidates. More importantly the balance between reactivity and stability of the reactive group was the focus point and the challenge in the design of the reactive building blocks. The first candidate consisted of thiazolidine masked...
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Novel fluorescent nucleotides for metabolic labelling and for the construction of DNA probes
Kuba, Miroslav ; Hocek, Michal (advisor) ; Slanina, Tomáš (referee) ; Míšek, Jiří (referee)
The aim of the thesis was to synthesize new nucleosides, nucleotides and the corresponding DNA probes bearing various fluorescent labels, which can be used for bioanalytical applications. In the first part of the thesis, 2'-deoxycytidine and the corresponding nucleoside triphosphate bearing tryptophan-based imidazolinone fluorophore were synthesized by Sonogashira cross-coupling reaction. The fluorophore showed sensitivity to pH and viscosity. Nucleotide was used for the construction of modified oligonucleotides (ON) and DNA by primer extension (PEX) or polymerase chain reaction (PCR). Labelled ON probe was used for sensing interaction with single-strand binding protein, which resulted in increased fluorescence intensity of modified ON. Next, thymidine and thymidine triphosphate labelled by bezylidene- tetrahydroxanthylium fluorophore were synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC). Fluorescence of the fluorophore is dependent on the polarity and viscosity of the environment. Incorporation of the modified nucleotide into DNA, by PEX or PCR, led to dramatic increase of the fluorescence presumably due to the interactions of the fluorophore in the major groove. Unfortunately, the modified nucleotide was not suitable for in cellulo imaging due to its cytotoxicity. The modified...
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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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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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