Interaction of genome-size DNA with phospholipid membrane in a cell-sized micro-watersphere as a model cellular system

Abstract

It has been established that a long DNA molecule (> 20–30 kilo base-pairs) exhibits a conformational transition from a coiled state to a highly folded state under the presence of condensing agents such as polyamines. We have investigated these changes in conformation of long DNA molecules at a single-molecule level by directly observing conformation of individual DNA molecules in the bulk aqueous solution under fluorescence microscopy. In a cell, DNA is placed in a micrometer-scale space surrounded by phospholipid membrane. In this study, to get insights into the structural characteristics of the genome-size DNA under such a cellular environmental condition, we encapsulated giant DNA (bacteriophage T4 DNA, 166 kilo base-pairs) labeled with fluorescent dyes in a cell-sized (20–60 μm) microsphere coated with phospholipid membrane, and investigated the conformational characteristics and distribution of the DNA in the microsphere under fluorescence microscopy. In the microsphere, T4 DNAs were diffusely distributed within the aqueous phase and exhibited a coiled conformation, when the microsphere was composed of eggPC (phosphatidylcholine from egg yolk). On the other hand, in the microsphere composed of DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), the DNAs were located on the membrane surface in the presence of high concentrations of Mg2+. Single-molecule observation of the DNAs with high-magnification images showed that the DNA exhibited an extended coil conformation and underwent the intra-molecular chain motion on the membrane surface. Under the same experimental condition, a short linear DNA (6 kilo base-pairs) was not bound to the DOPE membrane surface and was present in the aqueous phase in the microsphere, which suggests that the adsorption to the DOPE membrane surface in the presence of Mg2+ is a distinctive characteristic of long DNAs. These results are interpreted in terms of the structural characteristic and its roles of DNA in the cellular environment.