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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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