Akihiko Tsuji

Conformational transition of giant DNA in a confined space surrounded by a phospholipid membrane.

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.

ON-OFF switching of transcriptional activity of large DNA through a conformational transition in cooperation with phospholipid membrane.

We report that structural transitions of DNA cause the ON−OFF switching of transcriptional activity in cooperation with phospholipid membrane in a reconstituted artificial cell. It has been shown that long DNA of more than 20−30 kilo base-pairs exhibits a discrete conformational transition between a coiled state and highly folded states in aqueous solution, depending on the presence of various condensing agents such as polyamine. Recently, we reported a conformational transition of long DNA through interplay with phospholipid membrane, from a folded state in aqueous phase to an extended coil state on a membrane surface, in a cell-sized water-in-oil microdroplet covered by phosphatidylethanolamine monolayer (Kato, A.; Shindo, E.; Sakaue, T.; Tsuji, A.; Yoshikawa, K. Biophys. J.2009, 132, 1678−1686). In this study, to elucidate the effects of these conformational changes on the biologically important function of DNA, transcription, we investigated the transcriptional activity of DNA in a microdroplet. Transcriptional activity was evaluated at individual DNA molecule level by a method we developed, in which mRNA molecules are labeled with fluorescent oligonucleotide probes. Transcription proceeded on almost all of the DNA molecules with a coiled conformation in the aqueous phase. In the presence of a tetravalent amine, spermine, the DNA had a folded conformation, and transcription was completely inhibited. When the Mg2+ concentration was increased, DNA was adsorbed onto the inner surface of the membrane and exhibited an extended conformation. The transcription experiments showed that this conformational transition recovered transcriptional activity; transcription occurred on DNA molecules that were on the membrane.

Real-Time Monitoring of RNA Synthesis in a Phospholipid-Coated Microdroplet as a Live-Cell Model

We constructed a phospholipid‐coated water‐in‐oil microdroplet (20‐60 μm in diameter) that encapsulated plasmid DNA containing a human β‐actin cDNA sequence, fluorescent oligonucleotide probes, and other components of the transcription reaction. Transcription inside individual microdroplets was investigated in real time by using fluorescence microscopy. The progress of the transcription reaction was successively monitored by fluorescence resonance energy transfer (FRET), which was derived from the specific hybridization of fluorescent oligonucleotide probes to the β‐actin mRNAs synthesized. In microdroplets composed of phosphatidylethanolamine, DNAs were located in the aqueous phase or on the membrane surface depending on the MgII concentration. FRET images showed that transcription occurred on DNA in both states. Moreover, individual DNA molecules undergoing transcription were visualized as discrete FRET signals in cases in which small numbers of DNAs were present in the microdroplet.

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

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.