Artificial ribonucleases (ARs) are recognised as a potential strategy to selectively target and cleave biologically significant RNA in cells. However, in order to work as true enzymes they must exhibit catalytic turnover. Many of the reported ARs incorporate metal-containing centres (e.g. dysprosium, copper) in order to induce substantial phosphodiester cleavage, which is not amenable to use in vivo. Therefore, new strategic directions employing metal-independent ARs, such as peptidyl-oligonucleotide conjugates (POCs), need to be investigated. Previous work has shown that poor or non-complementary POCs demonstrate catalytic turnover of a HIV-1 substrate; however, sequence specificity is an issue. For POCs to be useful from a therapeutic standpoint they must only cleave specific RNA molecules and do so in a catalytic fashion, therefore removing the requirement for stoichiometric drug delivery and binding. Consequently, novel POC design strategies are required that allow selective RNA targeting but promote dissociation of the POC following phosphodiester cleavage. In this research, three types of different peptidyl-oligonucleotide conjugate designs have been implemented with the attempt to find an appropriate balance between selectivity and catalytic turnover.(i) Selective targeting and quantitative cleavage (97-100%) of a tRNAPhe target was achieved through phosphoramidate attachment of a 17-mer TΨC-targeting oligonucleotide to catalytic amphiphilic peptide sequences containing leucine, arginine and glycine. Although the half-life of tRNAPhe was less than 1 h on exposure to some of these POCs, hybridisation studies reveal that the POCs bind too tightly to their target RNA sequences and thus an excess of POC is required for efficient cleavage activity. The effect of peptide and oligonucleotide sequence variations as well as the role of enhanced conformational freedom via incorporating an abasic deoxyribose linker between the oligonucleotide targeting motif and catalytic peptide is also investigated. (ii) Most of ‘Dual’ peptidyl-oligonucleotide conjugates containing an amphiphilic RNA-cleaving peptide placed between two RNA recognition motifs directed towards the TΨC loop and 3’ acceptor stem of tRNAPhe demonstrate marked RNA binding and cleavage activities. Interestingly, those dual conjugates which showed poor or negligible binding ability in electrophoresis assays, demonstrated sufficient RNA cleavage (70%) within the vicinity of the 65GACAC61 target region. Therefore, weak POC:RNA complexes may exist which could facilitate substrate turnover. (iii) Finally, POCs were designed which induce bulge-loops in their target RNA region upon hybridisation. By introducing regions of non-complementarity into the oligonucleotide sequences, 2- to 5- membered bulges sizes were formed. Via attachment of a catalytic peptide to an internally modified oligonucleotide residue, catalytic peptides were placed directly adjacent to single-stranded RNA regions to promote cleavage by nuclease mimics. Through probing the hybridised complexes with RNase H, the presence of bulges were confirmed for all bulge-loop sizes, which will be followed by cleavage experiments to assess the possibility for reaction catalytic turnover. In conclusion, a variety of POCs have been synthesised, characterised and partially tested for their RNA cleaving and turnover activity. Based on the encouraging results presented POCs could be further developed to target disease specific RNA sequences such as micro- or messenger RNAs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677761 |
| Date | January 2015 |
| Creators | Williams, Aled |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/developing-a-new-generation-of-peptidyloligonucleotide-conjugates-with-desired-biocatalytic-properties(a53c3b27-bf03-47d6-b21c-47afb6e96018).html |
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