Self-Assembly in Phospholipid DNA - Protein Mixtures With Applications To Complex Formation in Cationic Liposome-Chromatin Systems

Abstract

We prepared complexes between cationic liposomes (CL) from a mixture of the neutral (DOPC) and the cationic (DOTAP) lipids with either nucleosome core particles (NCP) or chromatin arrays, prepared by in vitro over-expression. Microscopy showed the presence of distinct globules. Fluorescence labelling of the histone protein, DNA and lipid components showed complete co-localization under many conditions at the optical length scale, while separation of the histones from the DNA and lipids was sometimes found. Cryo-EM confirmed array aggregates with excess of positively charged lipid forming ordered complexes of multilamellar lipids. For complexes with lower cationic charge (50% DOTAP and 50% neutral DOPC), there is an indication of less order. In most Cryo-EM samples the complexes seems to have a “subunit size” on the order of 100-200 nm, consistent with DLS data. Synchrotron SAXS measurements were performed. The X-ray diffraction pattern demonstrates the lamellar Bragg peaks corresponding to an inter-lamellar separation of about 6-8 nm and sometimes presence of an in-plane DNA-DNA peak. This confirms a remarkably interesting phase behaviour for the system of DNA/protein (NCP or chromatin) with lipids. The SAXS and Cryo-TEM data clearly shows the formation of multilamellar aggregates with DNA sandwiched in between. This means that the DNA and the protein histone-octamer complex of the NCP and the chromatin arrays have dissociated. Hence, the question arises where the histone proteins are located, given the demonstrated co-localization at the length scale of the multilamellar complexes (a few hundred nm). One hypothesis is that the histone proteins are partially embedded in the bilayer and partially between the DNA domains. Alternatively, a few 50-100 nm size DNA-lipid complexes cluster, with histones associated in between, a type of associative phase separation.