A R T I C L E S Tsuji and Yoshikawa Figure 8. Effects of the Mg2+ and spermine concentrations on the number of discrete FRET signals formed by the transcription reaction in the bulk solution. The transcription reaction with λZAPII/actin DNA and ﬂuorescent oligonucleotide probes was run in the bulk solution for 30 min, and the reaction solution was then encapsulated in DOPE microdroplets. The numbers of discrete FRET signals were counted. Mg2+, which is considered to be due to the competitive binding of Mg2+ and spermine to the phosphate moieties of DNA.18 The results in Figure 8 are consistent with this fact when the results are interpreted to indicate that the conformational transition of λZAPII/actin DNA to a folded state occurred at a higher concentration of spermine in the presence of a higher concentration of Mg2+. We also performed experiments to estimate the amounts of RNA synthesized under the conditions of Figure 8 (15 mM Mg2+), which showed that RNA was not detected at more than 1.0 mM of spermine. These results show that all of the λZAPII/actin DNA molecules were “transcriptional- silent” (OFF) with 15 mM Mg2+ and 1.0 mM spermine in the bulk solution. We observed λZAPII/actin DNA in DOPE microdroplets at 15 mM Mg2+ and 1.0 mM spermine (Figure 7A(d-f)). They were located on the membrane surface and exhibited an extended conformation as in the absence of spermine, which shows that the conformational transition of λZAPII/actin DNA from a folded state in the bulk solution to an extended coil state on the phospholipid membrane surface takes place. When the transcription reaction was run, the discrete ﬂuorescence signals appeared on the membrane surface (Figure 7B(b)), which were very similar to those observed in the absence of spermine. These results show that transcription occurred under the condition in which it was completely inhibited in the bulk solution, when DNA was adsorbed to the PE membrane surface. Discussion In this study, the transcriptional activity of a long DNA in a cell-sized microdroplet coated with phospholipid membrane was investigated at individual DNA molecule level. The results are summarized in Figure 9. When the DNA was located in the aqueous phase with a coiled conformation, transcription was performed on the DNA (Figure 9a). In the presence of high concentrations of spermine, the DNA had a folded conformation in the aqueous phase. In this state, all of the DNA molecules were “transcriptional-silent” (Figure 9b). When the Mg2+ concentration increased, DNA was adsorbed onto the surface of the PE membrane and had an extended conformation. This conformational change of DNA through interplay with the phospholipid membrane was accompanied by the recovery of transcriptional activity (Figure 9c). We previously reported that T4 DNA is not adsorbed to a membrane surface composed of PC at a Mg2+ concentration at 12470 J. AM. CHEM. SOC. 9 VOL. 132, NO. 35, 2010 Figure 9. Summary of this study. which it is adsorbed onto PE membrane.18 We also observed that the adsorption of T4 DNA to the PE membrane surface does not occur when the microdroplet in which the DNA is encapsulated becomes large. This result is caused by the fact that the translational entropy loss of a DNA molecule upon adsorption increases as the microdroplet becomes larger, and that this entropy loss is not sufﬁciently compensated by the gain in free energy due to the DNA-phospholipid membrane interaction.18 Our observation in this study that is the ON-OFF switching of transcriptional activity of DNA through interplay with phospholipid membrane should depend on the phospholipid composition of the membrane and the volume of the micro- droplet in which DNA is conﬁned. In the experiments of gene expression, “transcriptional activity” has been usually evaluated in terms of amount of RNA synthesized from a large number of DNA molecules. However, there would be many cases that these estimates cannot provide the accurate information on the most essential matter regarding regulation of gene expression; whether the transcription of a speciﬁc gene on the DNA molecule is ON or OFF. Our method shown in this study, which directly evaluates whether or not the gene is transcribed at individual DNA molecule level, would provide very useful information for elucidating the character- istics of the regulation of gene expression. In this study, we demonstrated the ON-OFF switching of function (transcriptional activity) through the structural transi- tions of DNA. In a cell, genome DNA forms a self-assembled complex with histone proteins (nucleosome), which further forms highly packed structures. The structural changes in these DNA-protein complexes (chromatin) are considered to be responsible for the regulation of gene expression for a number of genes that are located on a proximity region of DNA.50,51 The regulation through structural changes/transitions is consid- ered to act in an all-or-none manner for all of the genes located in the region, and its characteristics are much different than those of the regulation of individual genes by the binding and release of some regulatory proteins to a speciﬁc sequence on DNA, when it works in a cell. Since there is only a single gene and several to several hundred regulatory proteins for the regulation of individual genes in a cell, the binding and release between a regulatory site of the gene and its binding proteins are under thermodynamic ﬂuctuation. In contrast, the structural changes are expected to provide the basis for more robust ON-OFF switching of transcription for a number of genes simulta- neously.52 The phenomena and experimental system shown in this study may be a good experimental model of the regulation (50) Gasser, S. M. Science 2002, 296, 1412–1416. (51) Horn, P. J.; Peterson, C. L. Science 2002, 297, 1824–1827. (52) Takenaka, Y.; Nagahara, H.; Kitahata, H.; Yoshikawa, K. Phys. ReV. E 2008, 77, 031905.