William Gaynor, James
(2008)
Incorporation of 3-s-phosphorothiolates into oligonucleotides : changing direction for RNA interference.
PhD thesis, University of Liverpool.
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Abstract
The main focus of this work falls into two sections. Firstly the synthesis and hybridisation studies of RNA duplexes containing 3'-S-phosphorothiolate (3'-sp) linkages, which are prepared using traditional automated 3'-5' solid-phase phosphoramidite chemistry. Secondly, investigations into the preparation of the 3'-sp linkage using a currently untried 5'-3', or reverse, synthesis approach. Both approaches have potential use in RNA interference (RNAi). For efficient RNAi activity, there are three key points; the small-interfering RNAs (siRNAs) must maintain an A-type helix; one terminus of the duplex is required to be more thermodynamically stable; and the strand whose 5' -end is less stably paired with the opposite strand is chosen as the guide strand. It has also been shown that RNAi activity does not totally rely on the 2' -hydroxyl functionality and can tolerate 2' -deoxy modifications. In terms of the sugar conformations, the 3' -sp linkage populates the RNA-like north conformation, and hence adopts an A-type helical conformation, to a greater extent than natural RNA, even though it lacks the 2' -hydroxyl group. Incorporating of a 3' -sp modifications into the uridine tract of the RNA duplex, 5'- r(GCGU1oGCG):5'-r(CGCA1QCGC),resulted in the thermal duplex stability rising by 1- 1.2 °C per modification. Therefore, by incorporating the 3' -sp linkage into either the 5' -end of the passenger strand, or the 3' -end of the guide strand, it is anticipated that the 3' -sp linkage can act as a compatible modification in RNAi. Using traditional 3'-5' synthesis, up to five 3'-sp linkages have been incorporated into the thymidine tract of 5'-d(GCGT1QGCG),each with a coupling efficiency of -93 %. By using reverse synthesis, it was hoped that the coupling yields could be enhanced so oligomers consisting entirely of 3'-sp modifications could be prepared; these oligomers could potentially be used as siRNA alternatives. We successfully incorporated a single 3'-sp modification into a DNA oligomer, so a proof of principle has been established. The yield of the thiol-phosphoramidite coupling is obviously poor and would need significantly enhancing for this technique to become a viable alternative to the traditionaI3'-5' synthesis. Optimisation studies are currently ongoing. As a small extra investigation, the possibility of synthesising 3'-thiopurine nucleosides was investigated using an enzymatic approach via transglycosilyation reactions. Initial studies indicate that 3'-thiothymidine and its corresponding 3'-thio-deoxyribose-1aphosphate derivative are substrates for thymidine phosphorylase and purine nucleoside phosphorylase, respectively.
| Item Type: | Thesis (PhD) |
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| Depositing User: | Symplectic Admin |
| Date Deposited: | 20 Oct 2023 09:24 |
| Last Modified: | 20 Oct 2023 09:43 |
| DOI: | 10.17638/03174430 |
| Copyright Statement: | Copyright © and Moral Rights for this thesis and any accompanying data (where applicable) are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge |
| URI: | https://livrepository.liverpool.ac.uk/id/eprint/3174430 |
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