by Zietlow, Christopher Mark

Abstract (Summary)
The enzymatic activity of DNase II has been investigated pertaining to non-viral gene therapy conditions that involve lipofectin, DMI-2, and poly-L-lysine. These compounds were included in DNase II digest assays of calf-thymus DNA and comparative kinetics calculated. Glycerol was included as a positive enzymatic effector. Three methods of detection were employed; UV, EPR, and gel electrophoresis. Comparative data of the three detection methods provided concurring evidence that low levels of lipofectin enhances DNA digestion by DNase II. EPR proved to be the most versatile method of detection for gene therapy conditions due to complex formations that cause turbidity and size constraint problems. The lipofectin-DNA complex formation has been investigated using spin-labeled DNA and EPR detection. The actual structure of the lipofectin-DNA complex remains uncertain. The structure of SL-DNA complexed to lipofectin and poly-L-lysine was investigated. The driving force of complex formation was verified to be electrostatically induced and maintained as evidenced by titration of SL-DNA with the cationic ligands, lipofectin and poly-L-lysine. The intensity of the subsequent EPR spectra decreased but the lineshapes remained. The SL-DNA was released from the complex with NaOH and NaCl recovering a majority of the EPR signal. The EPR signal of complexed spin-labeled calf-thymus double stranded DNA was found to behave differently than single stranded DNA with respect to release and signal intensity. Ascor
Bibliographical Information:


School:University of Cincinnati

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:non viral gene therapy epr spin labeled dna lipofection


Date of Publication:01/01/2001

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