去氧核醣核酸和PAMAM樹狀體錯合物之奈米結構研究

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研究生：	蘇群仁 Chun-Jen Su
論文名稱：	去氧核醣核酸和PAMAM樹狀體錯合物之奈米結構研究 Nanostructures of DNA-PAMAM Dendrimer Complexes: from Columnar Mesophase to Beads-on-String Structure
指導教授：	陳信龍 Hsin-Lung Chen
口試委員:
學位類別：	博士 Doctor
系所名稱：	工學院 - 化學工程學系 Department of Chemical Engineering
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	186
中文關鍵詞：	DNA 、錯合體、Poly(amido amine) 、DNA凝集、自組裝
外文關鍵詞：	DNA, Poly(amido amine), complex, self-assembly, nucleosome, chromatin, DNA condensation
相關次數：	點閱：2 下載：0
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Polyanionic DNA can bind electrostatically with cationic PAMAM dendrimer to form the complex exhibiting rich self-assembled structure at various length scales. This class of bioassembly has been considered as a non-viral gene delivery system for gene therapy and as a template or building block for DNA-based nanotechnology. Understanding the self-assembly behavior of DNA-dendrimer complex is crucial for the developments of both non-viral gene vectors and biomolecule-directed nanostructures.
This thesis presents a comprehensive study of the supramolecular structure of the complexes of DNA with poly(amidoamine) (PAMAM) dendrimers. The structure in pure water has been investigated as a function of dendrimer generation number, N/P ratio, temperature and dendrimer charge density controlled by the degree of protonation (dp). Here N/P ratio represents the molar ratio of the amine groups of dendrimer to the phosphate groups of DNA. For the complexes with low-generation dendrimers, i.e., G2 and G3, two types of ordered columnar mesophases, in which the DNA chains packed into long-range ordered lattice with in-plane hexagonal or square symmetry, were identified. The type of DNA packing was proposed to be governed by the interplay between DNA-dendrimer attraction and DNA-DNA repulsion. In general, hexagonal packing was the favorable structure for G2 complexes, while G3 system tended to form square phase. Smaller N/P ratio and larger dp could induce the formation of disordered columnar phase. As dp approached zero, a special hexagonal structure termed “H’ phase” formed to accommodate a large number of dendrimer within the hexagonal lattice.
For the complexes with G4 dendrimer, we observed the coexistence of two lattice orderings of both DNA and dendrimer components. Over a certain range of N/P ratio and dp, the DNA chains in the complexes were found to pack in the square lattice, and the dendrimer molecules situating in the interstitial tunnels further organized into a three-dimensional tetragonal lattice. The result indicated that interesting hierarchical nanostructure can be constructed by proper combination of building blocks with well-defined geometry, such as cylinder and sphere found in this study.
As the dendrimer generation was increased to nine, the complexation resulted in the beads-on-string structure found in DNA-histone complex constituting chromatin. At low dp, DNA chains wrapped around the dendrimer tightly with a distribution of pitches, and the chromatin-like fiber thus formed had a small persistent length due to weaker electrostatic repulsion. At dp = 0.5, the DNA chain wrapped around the dendrimer regularly and tightly with the pitch of ca. 2.6 nm. The resultant chromatin-like fiber was highly stiff with the persistent length probably close to several hundred nm.
Finally, a simple method for constructing a 2-D densely packed DNA nanostructure using the electrostatic complex of DNA with PAMAM G2 dendrimer was reported. Ordered DNA arrays were formed by drop-casting an aqueous solution containing positively overcharged complexes onto mica followed by a prolonged incubation. During the incubation, the complexes tended to adsorb onto the negatively charged mica surface through electrostatic attraction. The rodlike complexes organized to form ordered arrays to increase the surface density of the adsorbed complexes and hence the attractive free energy of adsorption.

ABSTRACT I
Table of Content III
List of Tables   VI
List of Figure   VII
Chapter
  1. Introduction and Literature Review
         1.1 Introduction 1
         1.2 Literature Review 5
             1.2.1 Introduction to the Starburst Poly(amidoamine) (PAMAM) Dendrimers 5
             1.2.1.1 Chemical structure 5
             1.2.1.2 Characteristics of PAMAM Dendrimers 9
                   I.  Topological Feature 9
                   II.  Degree of Protonation 15
             1.2.1.3 Bio-applications of PAMAM Dendrimers 17
         1.2.2 Characterization of DNA-histone Complexes 19
             1.2.2.1 Structure of Nucleosome 19
             1.2.2.2 Structure of Chromatin 24
         1.2.3 The Self-Assembly Behavior of DNA-dendrimer Complexes 32
       1.3 Overview of the Dissertation 43
       1.4 References 47
  2. Columnar Mesophases of the Complexes of DNA with Low-Generation Poly(amido amine) Dendrimers
      2.1 Introduction 51
      2.2 Experimental Section 53
        2.2.1 Materials 53
        2.2.2 Complex Preparations 54
        2.2.3 Zeta Potential Measurements 54
        2.2.4 Small Angle X-ray Scattering (SAXS) Measurements 54
        2.2.5 Transmission Electron Microscopy (TEM) Experiment 55
      2.3 Results and Discussion 55
         2.3.1 DNA-PAMAM G2 Complex 55
         2.3.2 DNA-PAMAM G3 Complex 81
      2.4 Conclusion 95
      2.5 References 97
  3. Double-Lattice Structure of the Complexes of DNA with Intermediate-Generation Dendrimer
     3.1 Introduction 99
     3.2 Experimental Section 100
        3.2.1 Materials 100
        3.2.2 Complex Preparations 101
        3.2.3 Small Angle X-ray Scattering (SAXS) Measurements 101
          3.2.4 Small Angle Neutron Scattering (SANS) Measurements 102
     3.3 Results and Discussion 102
     3.4 Conclusion 120
     3.5 References 121
 4. Beads-on-String Structure of the Complexes of DNA with High-Generation Dendrimer
    4.1 Introduction 123
    4.2 Experimental Section 126
       4.2.1 Materials 126
       4.2.2 Complex Preparations 126
       4.2.3 Small Angle X-ray Scattering (SAXS) Measurements 127
       4.2.4 Small Angle Neutron Scattering (SANS) Measurements 127
    4.3 Results and Discussion 128
    4.4 Conclusion 146
    4.5 References 147
 5. Two-Dimensional Densely Packed DNA Nanostructure Derived
     from DNA Ccomplexation with Low-Generation Poly(amidoamine) Dendrimer
    5.1 Introduction 149
    5.2 Experimental Section 150
    5.3 Results and Discussion 152
    5.4 Conclusions 158.
    5.5 Acknowledge 159
    5.6 References 160
Appendix 162
List of publication 185

                                

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