|
|
Enzymatic synthesis and study of properties of base-modified DNA
Viktorinová, Klára ; Hocek, Michal (advisor) ; Vrábel, Milan (referee)
The influence and specific interactions of chemical modifications in the DNA major groove have been systematically investigated for a long time. This work focuses on the enzymatic preparation of DNA bearing phenyl substituents on bases in milligram scale. For this synthesis, four 2'-deoxyribonucleoside triphosphates bearing an electron-rich phenyl group at position 7 of 7-deazapurines and at position 5 of pyrimidines (dAPhTP, dGPhTP, dCPhTP, dUPhTP) were prepared in prof. Hocek's group. The DNA polymerase KOD(exo-), which was expressed for the purpose of this work, was used to carry out the enzymatic synthesis. All four phenyl-modified 2'-deoxyribonucleoside triphosphates were successfully incorporated into the oligonucleotide chain using the primer extension and the DNA was synthesized in a milligram scale. The individual products were purified by dialysis and HPLC and their denaturation temperature was further monitored on UV-Vis spectrophotometer and their structure by spectroscopic method CD. The main objective of this and further extensive studies is mainly to understand the various biological processes and bioorthogonal reactions of macromolecules. [IN CZECH] Key words DNA, modifications, oligonucleotides, polymerases, PEX, PCR, structural analysis, CD
|
|
|
Photoaffinitive conjugation of biomolecules
Masárová, Petra ; Slanina, Tomáš (advisor) ; Vrábel, Milan (referee)
Photoclick chemistry represents a class of photo-activated click reactions generally characterised by precise spatial and temporal control, high selectivity, and quantitative yields. The following thesis will focus on several classes of light triggered click reactions and attempt to outline their chemistry in reversible nucleophilic addition, irreversible cycloaddition and Diels-Alder reactions initiated upon irradiation of photoactive precursors. The absorption of such precursors can be shifted towards visible light by derivatization of their chromophoric moieties, which allows for a non-harmful excitation in vivo and therefore offers promising prospects in biological applicability. The importance of click photochemistry resides in a remarkable selectivity that enables reactions to take place in highly complex, non-controlled biological environments. The photo-inducible precursors can be conjugated with various moieties of biological significance - a method most notably used in photoaffinity labelling. Considerable efforts have been made to explore the reactivity of photoactivatable compounds towards biological nucleophiles in various spheres of applied research which will all be discussed and summarised.
|
|
|
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...
|
|
|
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...
|
|
|
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...
|
|
|
Design and Synthesis of cell nucleus-targeted compounds for use in bioorthogonal reactions
Hrušák, Jáchym ; Vrábel, Milan (advisor) ; Míšek, Jiří (referee)
This bachelor thesis focuses on the design and synthesis of 1,2,4,5-tetrazine and trans- cyclooctene derivatives containing functional moieties which are able to target cellular nucleus and participate in bioorthogonal reactions. Based on literature research, the targeting moieties and the structure of the tetrazine and trans-cyclooctene derivatives were designed with the aim to achieve selective nuclear targeting and to ensure sufficient reactivity of the resulting conjugates under biological conditions. Selected candidates were then synthesized and their properties together with cellular reactivity were investigated in model experiments.
|
|
|
Hydrosolubilization of BODIPY for optical labelling of biomolecules
Bartoň, Jan ; Kotek, Jan (advisor) ; Vrábel, Milan (referee)
1 Abstract This work aims at showing synthesis and potential use of water-soluble fluorescent probes based on BODIPY. The preparation of probes containing bioorthogonal mono- and heterobifunctional functional groups was demonstrated. Ground work was done at the optimisation of reliable, scalable and fast sulfonation of BODIPY in 2,6-positions. A protocol for handling sulfonated BODIPY has been established; especially for the exchange of counterions. In counterion se- lection, their relation to synthetic pathway and biocompatibility were taken into consideration. The second part of the work shows series of water-soluble fluorescent probes, into which can be easily introduced bioactive or bioorthogonal functional groups. This can be used for click chemistry in connection with turn off/on probes or fluorescent sensing of molecules or ions. All this can be done in aqueous solution without organic solvents, which is relevant for biochemical, analytical and imaging applications. Keywords BODIPY, bifunctional, water-soluble, fluorescent probe, solubilization, biocompa- tible probes, bioorthogonal reaction, BODIPY sulfonation
|
|
|
Redox labelling of nucleic acids for analyzing nucleotide sequences and monitoring DNA-protein interactions
Fojta, Miroslav ; Havran, Luděk ; Horáková Brázdilová, Petra ; Pivoňková, Hana ; Kostečka, Pavel ; Macíčková-Cahová, Hana ; Raindlová, Veronika ; Vrábel, Milan ; Hocek, Michal
Nucleobase labelling of DNA for electrochemical sensing was attained through chemical modification of thymine bases with osmium tetroxide in the presence of nitrogenous ligands, or via enzymatic incorporation of nucleotide conjugates with redox-active moieties using labelled deoxynucleoside triphosphates. DNA hybridization, primer extension and PCR techniques were used for sequence-specific DNA assays. Tail-labelled DNA substrates were applied to monitor DNA binding by tumour suppressor p53 protein.
|
|
|
Příprava modifikovaných nukleosidů, nukleotidů a oligonukleotidů nesoucích komplexy kovů
Vrábel, Milan ; Hocek, Michal
Series of modified nucleosides and oligonucleotides bearing metal complexes were synthetized. Palladium catalyzed aqueous-phase cross-coupling reactions were used as key step in the synthesis of modified metallo-labeled nucleosides, nucleotides and oligonucleotides. The corresponding Fc modified dNTPs were good sustrates for DNA polymerases and were efficiently incorporated to DNA by primer extension (PEX). The modified nucleic acids are applicable as tools in bioanalysis.
|
|
| |
guest ::
