Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.580135
Title: Development of novel antitumour therapeutics based on DNA aptamers against the MUC1/Y protein
Author: Makwana , Vaidehi
Awarding Body: Open University
Current Institution: Open University
Date of Award: 2011
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Abstract:
Alterations in the expression of vital proteins of the cellular signalling pathways are at the forefront of molecular abnormalities established in cancer. Therefore, to understand the fundamental mechanisms of cancer initiation and progression, characterisation of tumour markers, such as key proteins like MUCIN that are highly expressed in malignant cells, is crucially important. Rapid drug discovery is a pressing need in the pharmaceutical industry. Agents that are able to bind tightly and selectively to the surface of diseased cells would greatly benefit both disease diagnosis and treatment. An aptamer is a functional oligonucleotide that has the appropriate sequence and structure to form a complex, possessing great affinity and selectivity for its target. Aptamers of high affinity and specificity have been successfully generated against the MUCIN splice variant from a non-conventional single round SELEX system termed SimpLex. The 72 base long aptamers were truncated to an ideal therapeutic length of 25 bases long via two different means. The first entails eliminating the primers and leaving only the central variable region and the second involves truncation of the aptamers based on their predicted secondary structure. Thu~, three aptamer versions were taken for further investigation, the complete and two truncated ones. However, as is it often the case, aptamers lack in vivo stability and are prone to rapid renal filtration and therefore require modification to achieve their full potential in vivo as therapeutic agents. Thus, the MUCIN selected aptamers were conjugated with a 32 kDa polyethylene glycol (pEG) polymer to increase their molecular weight to ea. 40 kDa, an appropriate size to avoid rapid renal clearance. The truncated aptamers, the pegylated aptamers and the full length aptamers were characterised employing qualitative techniques which, include EMSA, affinity column chromatography and DNA thermal denaturation studies. The aptamers were further assessed for their binding, specificity and internalisation by F ACs and fluorescence microscopy. The characterisation studies have shown that the anti-Ml.Cl/Y aptamers possess affinity and specificity for the MUCIfY protein and the conjugation of the aptamers to PEG has further enhanced their binding and internalisation potential. These encouraging results display potential future prospects as therapeutic agents for the pegylated aptamers and warrants their further future evaluation in in vivo studies.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.580135  DOI: Not available
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