Cooke, J.R.; (2005) Intrinsic DNA abnormalities and their effect on plasmid processing. Doctoral thesis , University of London.

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Abstract

Not all DNA has the familiar Watson-Crick, right-handed double helical structure. Left-handed helixes, triplexes, quadruplexes, bent DNA and regions of increased stiffness have all been encountered in nature. Some of these DNA structures are formed as a result of protein binding, while others are intrinsic. Such intrinsic structures may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. Intrinsic DNA structures were included at a defined point in a 2.9 kb plasmid, and their effects on cell growth rate, total plasmid yield, and topology (i.e. the relative proportions of supercoiled plasmid, open circular and linear forms), were determined. The stability of the inserted sequences was assessed using gel electrophoresis. Results suggest that Z-DNA is unstable in a batch Escherichia coli DH1 production system grown in complex medium. Encouragingly other sequences studied (triplex, bend and quadruplex) did not cause spontaneous deletions, and no detrimental effect was found on growth rate or on total plasmid yield indicating that such sequences could be included in future DNA products without any detrimental effect on plasmid yields. Although the intramolecular triplex studied significantly decreased the proportion of supercoiled species. The effect of different topological forms on transcription of a DNA vaccine in a cell free system was investigated, in order to determine the implications of this on specifications of plasmid products. Open circular plasmid was found to be expressed around 3.5 fold more than the supercoiled form. Current guidelines suggest that plasmid products should contain a minimum of 90% supercoiled species. The supercoiled form is not a single isoform, but contains plasmids with different numbers of supercoils (linking number). Measurement of the levels of supercoiled species in comparison to the other plasmid forms allows monitoring of the production process, however it is recommended that the guidelines be set on individual product basis i.e. drug efficacy will depend on delivery method, longevity and transcription levels.

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