Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.769595
Title: Peptide nucleic acid conjugates regulate gene expression in vitro : mode of action and new strategies for improved cellular uptake
Author: Vair-Turnbull, Douglas
ISNI:       0000 0004 7658 4444
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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Abstract:
RNA G-quadruplexes (G4) are an important class of nucleic acid secondary structures that are involved in mRNA translation, alternative splicing, localisation and 3'-end processing. Putative RNA G4 forming sequences have been identified in the regulatory regions of many disease-related genes, in particular oncogenes, and as a result have been of increasing interest as therapeutic targets for chemical intervention. G4-binding small molecule ligands and antisense oligonucleotides have both been used to effectively regulate translation. However, the therapeutic potential of drugs targeting RNA G4 structures is limited by a lack of specificity in the presence of alternative RNA secondary structures (including other G4s). Herein, the efficacy of a new generation of "sequence + structure" specific RNA G4 ligands is tested against the well characterised NRAS G4, chosen as a model system. Ligands were designed to target both (i) G4-specific structural features using a flat aromatic and cationic NDI platform and (ii) the single-stranded G4 flanking regions using short complementary peptide nucleic acid (PNA) sequences. Using an in vitro translation assay these PNA-small molecule conjugates were shown to inhibit translation with a significantly lower IC50 than the PNA or NDI alone. However, evidence suggested that the observed effect was mainly non-specific, which we suggest is due to electrostatic interactions mediated by the positively charged lysine residues added to the PNA-conjugates to improve solubility. In addition, a new 5'UTR G4 in the mRNA of the Aurora A kinase gene has been identified and characterised. Therapeutic PNA oligomers, built around a neutral peptide backbone instead of the negatively-charged oligonucleotide's sugar-phosphate backbone, offer a number of advantages when compared to natural oligonucleotides, including resistance to proteases and higher binding affinity and specificity for complementary DNA (or RNA) sequences. However, PNA oligomers have very poor membrane permeability, which means that PNA conjugation is likely to significantly decrease their bioavailability. Conjugation of molecular transporters, such as cell penetrating peptides (or peptoids), is currently the most viable method to improve cellular uptake of therapeutic PNA oligomers. Here we report on the development of an in vitro assay and present initial results showing that sonoporation (formation of small pores in cell membranes by combined use of ultrasound and microbubbles) can be used to increase the cellular uptake of PNAs without the need for pre-conjugation to cell-penetrating peptides.
Supervisor: Ladame, Sylvain Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.769595  DOI:
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