Cancer is a leading cause of death worldwide, accounting for around 13% of all death [1]. Traditional cancer therapeutics usually require careful selection of one or more intervention, such as surgery, radiotherapy, and chemotherapy, which have made momentous progress, but have ample limitations [2]. The next generation of cancer therapeutics will specifically target processes responsible for the growth and survival of cancer cells. Among the most promising of these molecularly-targeted therapeutics are short interfering RNAs (siRNAs). These siRNAs serve as the effectors of RNA interference, a naturally occurring and highly specific mechanism for regulating gene expression through sequence-specific degradation of messenger RNA. However, the native structure of RNA is plagued with undesirable chemical properties. For example, the sugar-phosphate backbone contains a negative charge which hinders its ability to cross the negatively charged lipid bilayer. Furthermore, the phosphodiester backbone is a substrate for nucleases, which catalytically cleaves the phosphate-oxygen bond, thus degrading the native RNA [3]. As such, there is widespread interest in chemically modifying the backbone of siRNAs in order to overcome some of the inherent problems with its native structure.
There have been only two reports that have employed amide-bond linkages as phosphate replacements within siRNAs [4, 5]. In both of these studies, the amide bond containing monomer units were placed at the 3’-overhangs and not within the internal Watson-Crick region of the double stranded siRNA due to the limitation of standard solid-phase oligonucleotide synthesis. In this thesis, we proposed to utilize phosphoramidite chemistry to localize internal amide-bond modifications [6]. A practical synthesis of a peptide nucleic acid unit combined with an RNA nucleoside (PNA-RNA dimer, UaU) is reported [7]. Using this PNA-RNA dimer phosphoramidite allows us to control the site-specific location of the internal amide-bond
modification throughout the desired RNA strand. Polyacrylamide gel (PAGE) and mass spectrometry analysis were performed to ensure the formation of full-length modified siRNA molecules.
The effects of these modifications were explored with respect to the biophysical and biological properties of the modified siRNAs. The techniques used in this work included hybridization affinity assays (melting temperature), secondary structure determination (circular dichroism), cell-based luciferase assays, and nuclease stability assays. Melting temperature experiment reveals that localizing a UaU dimer unit within the RNA oligonucleotides has an overall destabilizing effect, whereas UaU modifications at the 3’-overhang positions show little change in thermal stability. Circular dichroism experimental results illustrate that all chemically modified siRNAs exhibit the standard A-form helix. In cell-based luciferase assays, we utilized two different target sequences and our results highlight the compatibility of utilizing a neutral amide-bond backbone within siRNAs. Specifically, the internal amide-bond modification is compatible within the RNAi machinery when placed at 3’-overhang position in the sense strand of the double-stranded siRNA. However, poor efficacy is observed when this unit is placed adjacent the Ago 2 cleavage site on the antisense strand. The nuclease stability assays reveal that the introduction of a PNA-RNA dimer at the 3’-end of the siRNA where the exonuclease cleaves the terminal nucleotide, increased markedly the resistance to serum-derived nucleases. To the best of our knowledge, this is the first report that involves amide-bonds as phosphate backbone replacements within the internal regions of siRNAs and thus opens the future possibility for examining and utilizing this modification in studying new structure-function relationships. / UOIT
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OOSHDU.10155/300 |
| Date | 18 January 2013 |
| Creators | Gong, Wei |
| Contributors | Desaulniers, jean-Paul |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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