INTERACTION OF DNA WITH CATIONIC VESICLES FROM AMPHIPHILIC METAL COMPLEXES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。据报道，阳离子脂质体促进DNA跨细胞膜转移，被认为是基因治疗的潜在非病毒DNA载体。高分辨率同步x射线衍射（XRD）研究表明，脂质体基因转染效率与DNA与阳离子囊泡的相互作用机制有关。为了合理设计一种既能通过静电力又能通过氢键与DNA相互作用的阳离子脂质体，我们合成了一种新型的两亲性金属配合物，这种配合物能够在具有高阳离子电荷密度和明确的氢键基序的水介质中形成囊泡，适合与DNA的磷酸主基结合。新形成的囊泡已被证明可以有效地将绿色荧光蛋白（GFP） pEGFP-N1 （4.9 kb）转染到哺乳动物HEK 293-T细胞系中。动态光散射（DLS）研究表明，当脂质体与线性DNA络合时，其直径从400 nm增加到800 nm。通过透射电子显微镜（TEM）进行的研究表明，阳离子脂质体与DNA相互作用形成洋葱状结构，其中DNA单层夹在阳离子脂质双层之间。为了证实这一观察结果，我们建议使用小角度x射线散射（SAXS），我们期望看到插入DNA层的布拉格反射结果。研究合成的阳离子脂质体与DNA的相互作用，有助于建立脂质体辅助细胞转染机制的新理论。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cationic liposomes had been reported to facilitate DNA transfer across cell membranes and are considered to be potential non-viral DNA carriers for gene therapy. High-resolution synchrotron x-ray diffraction (XRD) studies show that the efficiency of the liposome gene transfection is associated with the interaction mechanism between DNA and the cationic vesicles. In an effort to rationally design a cationic liposome that could interact with DNA not only by electrostatic forces but by hydrogen bonding, we synthesized novel amphiphilic metal complexes which were able to form vesicles in aqueous media with high cationic charge densities and well-defined hydrogen bonding motifs suitable for binding to the phosphate backbone groups of DNA. The newly formed vesicles have been proved effective for the transfection of the Green Fluorescent Protein (GFP) pEGFP-N1 (4.9 kb) into mammalian HEK 293-T cell lines. Dynamic light scattering (DLS) studies show a liposome diameter increase from 400 nm to 800 nm when they are complexed with linear DNA. Studies done by transmission electron microscopy (TEM) suggest that the cationic liposome interacts with DNA forming an onion-like structure where DNA monolayers are sandwiched between cationic lipid bilayers. In order to confirm this observation we propose to use small angle x-ray scattering (SAXS) where we expect to see the Bragg reflections result from the intercalated DNA layer. The study of the interactions between DNA and the synthetic cationic liposome could help to develop a new theory on the mechanism of liposome assisted cell transfection.