ABSTRACT
Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.
Subject(s)
DNA, Superhelical/analysis , Scattering, Radiation , DNA Damage , DNA, Superhelical/radiation effects , Electrophoresis, Agar Gel , Energy Transfer , Methods , Nucleic Acid Conformation , Particle SizeABSTRACT
The development of non-viral vectors for gene delivery has primarily focused on improving the efficiency of gene transfer in vivo. Although there is clearly a need to improve delivery efficiency, studies also indicate that the physical stability of non-viral vectors is not nearly adequate for a marketable pharmaceutical product. Here, we describe the different strategies that have been used to enhance stability and discuss the mechanisms by which prolonged stabilization (>2 years) might be achieved.
