It has been established that a long DNA molecule exhibits a large discrete conformational change from a coiled state to a highly folded state in aqueous solution, depending on the presence of various condensing agents such as polyamines. In this study, T4 DNA labeled with fluorescent dyes was encapsulated in a cell-sized microdroplet covered with a phospholipid membrane to investigate the conformational behavior of a DNA molecule in such a confined space. Fluorescence microscopy showed that the presence of Mg(2+) induced the adsorption of DNA onto the membrane inner-surface of a droplet composed of phosphatidylethanolamine, while no adsorption was observed onto a phosphatidylcholine membrane. Under the presence of spermine (tetravalent amine), DNA had a folded conformation in the bulk solution. However, when these molecules were encapsulated in the microdroplet, DNA adsorbed onto the membrane surface accompanied by unfolding of its structure into an extended coil conformation under high concentrations of Mg(2+). In addition, DNA molecules trapped in large droplets tended not to be adsorbed on the membrane, i.e., no conformational transition occurred. A thermodynamic analysis suggests that the translational entropy loss of a DNA molecule that is accompanied by adsorption is a key factor in these phenomena under micrometer-scale confinement.

We developed a novel method for the in situ analysis of the higher-order structure of an individual genome from a single Escherichia coli cell using laser tweezers. Initially, condensed DNA was stably grasped by a laser without any chemical modification and without physical attachment to an artificial object such as micro-plastic beads. Under optical transport, the trapped genome gradually unfolded in solution due to viscous friction. Interestingly, the nucleoid DNA from a log-phase cell is almost fully elongated, whereas in the stationary phase, unfolding of the nucleoid is characterized by step-wise elongation of 1.7-5.1 microm, corresponding to a size of 5-15 kbp, and a few tightly packed domains remain along the DNA chain. This suggests the coexistence of tightly packed and swollen domains in the genome in the stationary phase.

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