On trifluoromethylated RNA and the twister ribozyme / Marija Košutić
VerfasserKošutić, Marija
Betreuer / BetreuerinnenMicura, Ronald
Umfang124 S. : zahlr. Ill., graph. Darst.
HochschulschriftInnsbruck, Univ., Diss., 2015
Datum der AbgabeMai 2015
Schlagwörter (DE)trifluoromethylated RNA / twister ribozyme
Schlagwörter (GND)RNS / Trifluormethylierung / Ribozym
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Zusammenfassung (Deutsch)

A wide range of natural RNAs carry complex nucleoside modifications which arise from biosynthetic transformations during the RNA maturation pathway. Very often the cellular function of these modifications is far from being well understood. In most cases, they significantly change the biochemical, and physicochemical properties in comparison to the unmodified RNA. Unfortunately, a major hurdle for their thorough investigation is their low abundance. In general, only very small amounts of the modified RNAs and the corresponding modified nucleosides are accessible from natural sources. Therefore, synthetic chemists are challenged to develop efficient routes towards the corresponding phosphoramidite building blocks for RNA solid-phase synthesis and to push the size limitation of currently about 60 nt length to larger synthetic RNAs.

In this context, considerable attention is given towards the development of artificially modified oligonucleotides pointing out the significance of chemically modified nucleosides and their site-specific incorporation into RNA. It was demonstrated that RNAs bearing non-natural modifications have many potential uses in basic and applied research, ranging from RNA imaging and localization, structure and dynamics studies to applications in oligonucleotide therapeutics. Driven by that observation, the research, presented in the first chapter of this thesis, was focused on the design, synthesis and characterization of labeled nucleosides and nucleic acid constructs, and the evaluation of their structural and biology-related properties by modern spectroscopic methods. Specifically, my study has been focused on the development of novel 2-trifluoromethylthio-labeled RNA derivatives as markers in 19F NMR of biomolecular systems.

The second part of this thesis is concerned with nucleolytic ribozymes. These RNAs are known to play key roles in cellular processes because of their ability to catalyze site-specific cleavage and ligation of its own backbone. However, there are still many open questions regarding the formation of ribozymes active structures, their structural dynamics, and the precise biocatalytic mechanisms. Recently, the discovery of the new class of self-cleaving ribozyme motifs, named twister, triggered severe efforts of several research groups worldwide to explore its structure and mode of action. In this context, I have synthesized a variety of chemically modified twister ribozymes presented in second chapter of the thesis. These derivatives made it possible to solve the high-resolution structure through X-ray crystallography in collaboration with the group of Dinshaw Patel. Additionally, detailed investigations on the cleavage activities and kinetic measurements of the synthesized twister variants with single-site modifications contributed substantially to elucidate this ribozymes catalytic mechanism.

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