title: Impact of intrinsic DNA structure on processing of plasmids for gene therapy and DNA vaccines.
abstract: Several non-Watson Crick DNA structures have been discovered to date, which may be incorporated into future plasmid constructs for gene therapy and DNA vaccine products. In this study, 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 were assessed using gel electrophoresis. Z-DNA was shown to be 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 intra molecular triplex studied significantly decreased the proportion of supercoiled species.
authors.affiliation: Department of Biochemical Engineering, UCL, Torrington Place, London WC1E 7JE, UK.
authors.forename: James R
authors.initials: JR
authors.lastname: Cooke
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: McKie
authors.forename: John M
authors.initials: JM
authors.lastname: Ward
authors.forename: Eli
authors.initials: E
authors.lastname: Keshavarz-Moore
mesh_terms: D001483:Base Sequence; D019149:Bioreactors; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry*; D043542:DNA, Z-Form / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D015316:Genetic Therapy / Q000379:methods; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D010982:Platelet-Derived Growth Factor / Q000235:genetics; D019444:Vaccines, DNA / Q000235:genetics
chemical_list: D043542:DNA, Z-Form; D010982:Platelet-Derived Growth Factor; D019444:Vaccines, DNA; D004247:DNA