Three-Dimensional Structural Analysis of Lipid and DNA Complex using Zernike Phase Contrast Transmission Electron Microscope Tomography

Lipids and DNA complex, so called lipoplex, is a promising materials for gene (for example si-RNA) delivery. The lipoplex were thought to be randomly agglomerated. But the first article written by C. R. Safinya et al. (Science 1998, 128, 78-81) suggested an model of orderly aligned lamellar layers proved by small angle x-ray scattering (SAXS).In this study, we observe the lipoplex by transmission electron microscope (TEM). Samples are the lipoplex made from cationic lipid: DOTAP (1,2-Dioleoyl-3-Trimethylammonium-Propane), zwitter ionic lipid: DOPC (1,2-Doleoyl-sn-Glycero-3-Phosphocholine), and plasmid DNA. Newly developed Zernike phase contrast TEM is used in order to get more contrast, and cryo-transfer is used in order to preserve configuration of bio matter into ice by the quick freezing.In addition, we use a new observation method; TEM tomography. If we use transmission images from conventional TEM, it is difficult to know the conformational structures of complicated arranged object. To solve this problem, we take dozens of TEM images with be tilted from −70 to 70 degree by 2 degree steps, and reconstructed to the conformational structure.Due to the tomography treatment, the conformational structure of the complex can be analyzed. We can analyze not only the boundary structure of the complex agglomeration, but also inside structure. Moreover, we want to discuss about the production process of the lamellar structure based on the results of the TEM tomography observation.