Study of DNA condensation for applications in nucleosome interactions and gene delivery with novel peptide vehicles

• Main Author: Huang, Dandan
• Other Authors: James P. Tam
• Format: Theses and Dissertations
• Language: English
• Published: 2009
• Subjects: DRNTU::Science::Biological sciences::Human anatomy and physiology::Deoxyribonucleic acids
• Online Access: https://hdl.handle.net/10356/15165
• Institution: Nanyang Technological University

The basic idea of gene therapy is to replace abnormal genes with normal genes. To realize this replacement, carrier molecules called gene delivery vectors are needed. The shortcomings of viral gene delivery vectors have stimulated the development of nonviral vectors. In this work, a series of novel ε-polylysines with different chain length and side chains have been designed and characterized to be used as gene delivery agents. I first physicochemically characterized DNA compaction by these novel ε-polylysines with Dynamic Light Scattering (DLS), Fluorescence Microscopy (FM) and Isothermal Titration Calorimetry (ITC). The ε-polylysines gave typical titration curves when added into DNA solutions. EC50 (the charge concentration of peptide at the midpoint of the scattering intensity curve) increased with the increase of ε-polylysine charge, indicating the electrostatic interactions between negatively charged DNA and positively charged ε-polylysines. For low charge ε-polylysines (n=5~7), EC50 increased as salt concentration increased in the solution. Among ε-K10 homologues with different side chains, ε-(LYR)K10 is the most efficient DNA compaction agent. EC50 of both ε-K10 and ε-(LYR)K10 increased with increase of the pH of the solution. With fluorescence microscopy, the free DNA molecules in extended coils and compacted DNA were clearly observed. The ITC results confirmed the behavior of DNA compaction. I have also conducted studies of nucleosome core particles (NCP) aggregation using precipitation assay and DLS. NCP is the most regular central part of the nucleosome on which DNA mainly exists in the nucleus of eukaryotic cells. NCP precipitation curves were obtained with different cations, Mg2+, Ca2+, K+ and Na+ respectively and they are consistent with reported work. HMGNs (high mobility group N) are a family of proteins found in the nuclei of all mammals and most vertebrate cells. Mg2+ or Spm4+ (cationic form of spermine) was used to test the effect of HMGN2 and its truncated form XN2 [fragment of HMGN2 (a.a. from 17 to 46)] on NCP aggregation. The results are rather complicated and different for different conditions. Therefore, more experiment need to be done to further explain the results.