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Title: Development of site-specific RNA labeling strategies to probe alternative RNA splicing
Author: Kosmidis, Tilemachos D.
ISNI:       0000 0004 6348 134X
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2016
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The objective of this project was to develop a method for the labeling of RNA at specific locations using non-natural base pairs. This thesis details the efforts made towards this objective through the development of a modular synthetic platform for an expanded genetic alphabet and its evaluation using transcriptional assays. Chapter 1 introduces the structure and function of RNA, followed by a description of RNA processing events with emphasis placed on alternative RNA splicing and how aberrations in splicing can lead to disease. The concept of RNA labeling follows and examples from the literature are presented along with challenges associated with these techniques to elucidate key mechanistic splicing concepts. The concept of orthogonal base pairs is then introduced along with representative examples from the literature. The application of this concept in RNA labeling is then presented along with key examples from the literature. The limitations of these methods are highlighted followed by the specific aims behind this project. Chapter 2 presents the efforts made towards the development of a modular synthetic route for the synthesis of C-ribonucleosides, with a Heck reaction as the key step. Specifically, its application in the synthesis of the Z ribonucleoside developed by the Benner group is investigated. The chapter starts with re-introducing the Z/P pair developed by the Benner group and highlighting the reasons that placed it as our chosen base pair for RNA labeling. The synthesis of C-deoxyribonucleosides utilizing the Heck reaction is presented, followed by the challenges associated with C-ribonucleoside synthesis. The syntheses of the alkene and halide coupling partners of the proposed Heck reaction are firstly presented. Investigations on the Heck reaction are then presented, followed by investigations on the transformation of nitro group in Z to moieties suitable for further derivatization. The key outcome of this work is that a synthetic route involving the Heck reaction for the synthesis of C-ribonucleosidesis not feasible. This is due to long reaction times required to drive the Heck reaction to completion and difficulties encountered with elucidation of its stereselectivity. Furthermore, PCR experiments conducted by our collaborators within the Eperon group at the University of Leicester revealed significant misincorporation of Z opposite G. Consequently, a change in strategy towards the use of nucleotides developed by the Hirao group was made. Chapter 3 describes the development of a robust synthetic route for the synthesis of a library of C6-functionalized 2-aminopurines as potential candidates for an expanded genetic alphabet in RNA. The s/Pa pair developed by the Hirao group is re-introduced. The installation of an alkyne functionality on the guanosine scaffold via a Sonogashira reaction is described,followed by investigations of [3+2] cycloaddition reactions between the alkyne and azides or aldehyde oximes. Finally, the development of a novel Suzuki-Miyaura protocol for the direct installation of heterocyclic substituents on the guanosine scaffold is also reported. Key outcomes of this chapter are the following. A robust method was developed for the expedient synthesis of C6-functionalized s analogues. This method enabled access to various classes of analogues in three or six steps,including triazoles, isoxazoles, thiophenes and pyrazoles. Attempts to install an azide moiety to the guanosine were partly successful, but the strategy was abanonded due to reaction reproducibility issues. In addition, a C-H activation strategy was not successful on installing an oxazole moiety. Chapter 4 details the efforts towards the synthesis of nucleoside triphosphates based on the s analogues described in Chapter 3. This chapter will begin with the presentation of the most common strategies for the synthesis of nucleoside triphosphates. Initial attempts to synthesize triphosphates by global deprotection of nucleosides synthesized in Chapter 3 are next described. Attempts to install a silyl ether group in the 5’-OH are presented, followed by the installation of acetates on the 2’/3’ hydroxyls and attempts to protect the exocyclic amine on guanosine as the phenoxyacetamide. The installation of a pyrazole moiety via Suzuki-Miyauraprotocol using the corresponding boronic acid pinacol ester described in Chapter 3 is presented. The installation of an alkyne moiety is also described and the synthesis of an isoxazole analogue using this alkyne precursor will follow. The chapter will end with the presentation of the synthesis of pyrazole and isoxazole triphosphate analogues. The key outcome of this chapter is that employing different protecting groups on the 5’ and the 2’/3’ hydroxyls enabled the synthesis of pure triphosphates. Chapter 5 presents the evaluation of the nucleotide triphosphates synthesized in Chapter 4 regarding their transcriptional efficiency. Key outcome of this work are that high concentrations ( > 0.5 mM mM) are needed in order to observe significant incorporation of the analogues, compared to s, which needs 0.1 mM for efficient incorporation. At high concentration, the isoxazole moiety exhibits better incorporation efficiency compared to the pyrazole analogue. Furthermore, the addition of 0.5 mM of MnCl2 resulted in increased incorporation efficiency of the pyrazole analogue, while s and the isoxazole exhibited reduced efficiency under these conditions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral