Inaktivierung von mRNAs und miRNAs durch DNA/LNA-basierte und mit zusätzlichen Funktionalitäten konjugierte Antisense-Oligonukleotide

Seit der Entdeckung der RNA Interferenz (RNAi) Ende der 90er Jahre wurde dieser Mechanismus als gentechnisches Werkzeug stetig weiterentwickelt und in den verschiedensten biologischen sowie medizinischen Bereichen eingesetzt. Dies ermöglichte nicht nur die Aufklärung unbekannter Gen-Funktionen durch...

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Bibliographic Details
Main Author: Thomas, Laura
Contributors: Hartmann, Roland K., (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
Online Access:PDF Full Text
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Since the discovery of RNA Interference (RNAi) in the late 90s, ongoing work and development of this genetic engineering tool has been executed in different biological and medical fields. This enabled the discovery of unknown protein functions and the development of RNA-Interference-based therapeutic agents. Usage of small non-coding RNAs, which can regulate the gene expression via RNAi, is one very promising therapeutic option, because microRNAs (miRNAs) possess a substantial influence on the regulation of countless biological processes such as differentiation, or cell proliferation. Therefore they can have tumour-suppressive as well as oncogenic properties. Besides innate miRNAs, small synthetic RNAs have been developed for therapeutical approaches and genetic analysis such as small interfering RNAs (siRNAs), enabling the usage of more stabilized and modified RNAs. The possibility of modification increased the areas of application for miRNAs and siRNAs as well as facilitating new therapeutic options. The first project of this thesis analysed the possibility of redirecting RISC to a specific mRNA via a bifunctional adapter oligonucleotide. This approach allows an inhibition of oncogenic miRNA function and a simultaneous suppression of a proto-oncogenic mRNA translation, which can lead to a synergistic antitumor effect. One adapter part is thereby directed against an oncogenic miRNA, which was loaded bevor into a RISC. The 3'-UTR of a specific mRNA is addressed by the second part of the adapter, which enables then the redirection of the indirect bound RISC to the addressed mRNA and thereafter inducing its degradation. At the beginning of this analysis the proto-oncogen PIM1 and the oncogenic miR-20a were addressed via the bifunctional adapter. Within in vitro studies it was shown that it is possible to bind and isolate miR-20a loaded RISCs from cell lysates. The bifunctional adapter effect was then examined in cell culture experiments, in which the protein expression of the proto-oncogene PIM1 and the tumour-suppressor P21, which is a target of miR-20a, was analysed. In observed cases the cell culture experiments showed a clear effect on the PIM1 expression, however this could not be reproduced stably. A change of the addressing miRNA part from miR-20a to the complete miR-17a family and then to the higher expressed let-7 miRNA family was also not able to stabilize the influence on the PIM1 and P21 expression. Nevertheless, the group of Michael Göbel was recently able to show a functional redirection of RISCs in in vitro assays. This indicates that in cell culture experiments more constant results are possible with a more stable mRNA system. The second topic of this thesis addressed the function of a metal-free synthetic nuclease-conjugate with a DNA/LNA mixmer-oligonucleotide and tris(2-aminobenzimidazole). Building up on very promising results from the Göbel group with DNA and PNA conjugates, in this thesis in vitro cleavage kinetics were done with a DNA/LNA mixmer conjugate. Those confirmed a clear reduction of the RNA substrate half-life compared to former analysed conjugates and furthermore a specific and efficient cleavage of longer RNA substrates with a length up to 412 nt. The cleavage position could be characterised and localized around the binding of the mixmer oligonucleotide with the addressed RNA substrate. The PIM1 mRNA was then addressed in subsequent cell culture experiments, in which occasional effects were observed, yet not reproducible. The promising in vitro results nevertheless indicate, that with further improvement of the cleavage kinetics, clear cell culture effects can be obtained.