Extensive investigations have been made to try and understand the physical properties and structures of condensed DNA phases in vitro during the past decades (Bloomfield, 1991, Marquet and Houssier, 1991, Bloomfield, 1996). The packing pathways of DNA molecules are three dimensional processes, and are not yet fully understood (Yoshikawa et al., 1997). Distinguishing different single DNA molecules at different locales in time in the presence of condensating or dissociating agents is crucial for understanding the mechanisms of packing and unpacking of DNA molecules. The aim of this study is to provide an increased understanding of the some of the pathways of packing and unpacking of DNA. This aim was achieved by monitoring in time and at molecular scale the interaction between the DNA and polyamidoamine dendrimers, as condensating poly cations, and by observing the dissociation of some of these condensates in time when they were exposed to DNase I enzyme, as a dissociating in vivo agent. The main techniques used were atomic force microscopy (AFM) and gel electrophoresis. We believe that the results could be beneficial to the understanding of the in vivo condensation and dissociation process of certain DNA morphologies. Chapter 1 will focus on providing an overview of the single molecule techniques used, atomic force microscopy as a means of detecting individual biomolecules in near physiological conditions with time and its application in monitoring non-viral gene delivery systems on surfaces. Methods used for gene delivery, and the PAMAM dendrimers as one of the recently applied polymer in non-viral gene delivery are also reviewed. The materials and methods used in this thesis were considered in chapter 2. Chapter 3 will concentrate on the factors effecting the interactions of generation 4, 6 and 8 PAMAM dendrimers on surfaces. An understanding of these interfacial interactions is important to understand their effects on the individual DNA molecules. This aim was achieved by using AFM as an imaging and force measuring tool to visualize and characterize the adsorption of these dendrimers on mica, gold and on alkanethiol self assembled monolayers (SAMs). Developing a deep understanding of the adsorption of DNA onto oppositely charged substrates would be of fundamental importance in understand the packing and unpacking pathways of these molecules. This philosophy is demonstrated in Chapter 4 in which the ability of the monovalent cations to facilitate imaging of DNA, and the effect of these monovalent cations in the partial condensation of DNA is explored. In addition, Chapter 4 introduces DNA imaging in the presence of divalent cations in liquid and in air. The folding pathways of dendrimer-induced DNA condensation with time on the surface of mica in aqueous environment were the targets of Chapter 5. In addition, the surface-influenced DNA condensation in the presence and absence of sufficient soluble cations and the ionic strength dependence were also studied. Structural volume and hence information regarding the number of plasmid molecules in each condensate was explored. Furthermore, the effect of loading ratio and generation type on the complex retardation in gel electrophoresis was investigated. Chapter 6 investigates the different mechanisms of the DNA-PAMAM dendrimer condensate relaxation and fragmentation by DNase I with time and explores the mechanisms of wrapping and unwrapping of the DNA on the larger generations of dendrimers. The final chapter, Chapter 7, discuses the progress made towards the aims of this thesis. Interestingly, this investigation is one of the first to apply atomic force microscopy operating in liquid to visualize at the molecular scale and in real time DNA molecules in the absence of multivalent cations, to explore the formation of DNA complexes with ethylene di amine PAMAM dendrimers that have real potential as gene delivery vectors and to investigate the different condensation and dissociation pathways of individual DNA molecules. Overall it is hoped that the work described in this thesis provides a step forward in the methods applied for AFM based nano-force biomolecular imaging with time and provides a valuable information that aid in developing a successful non-viral gene delivery system.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:514609 |
| Date | January 2004 |
| Creators | Gharib Abdelhady, Hosam |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://eprints.nottingham.ac.uk/10077/ |
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