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Title: Specific detection of 14-3-3 proteins by ssDNA aptamers
Author: Stevenson, Ross
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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A library of ssDNA aptamers has been screened and a number of molecules which show high binding affinity to 14-3-3 proteins have been isolated. The gene encoding mammalian 14-3-3 γ modified by (his)6 tagging, was overexpressed in E. coli and purified to homogeneity. The recombinant 14-3-3 γ was characterised using a series of techniques ensuring that the expressed protein had similar physical and immunological characteristics to that of wild type 14-3-3 γ. An ssDNA aptamer library was synthesised and aptamers selected following an adapted SELEX (systematic evolution of ligands by exponential enrichment) protocol. After 16 selection rounds, aptamers contained within the enriched pool were cloned and sequenced for further analysis. A sample of twenty of the sequenced aptamers were synthesised and biotinylated and their interaction with (his)614-3-3γ was analysed by surface plasmon resonance and by enzyme linked oligonucleotide assay (ELONA). The two aptamers showing the greatest association to (his)614-3-3γ were further analysed against all 14-3-3 isoforms using ELONA and by dot-blot analysis. The high affinity aptamer, S16-8, was used to affinity purify recombinant 14-3-3 spiked into ovine serum albumin and wild type 14-3-3 in mouse brain homogenate and to detect and concentrate 14-3-3 protein in a scrapie positive cerebrospinal fluid (CSF) sample. The results of this work suggest that this aptamer could be used as a probe to detect isoforms of wild-type 14-3-3 proteins from a range of biological fluids.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available