ON-OFF Switching of Long DNA Transcription A R T I C L E S Figure 1. Experimental system. (A) Long DNA molecule has three states in a cell-sized water-in-oil microdroplet coated with phospholipid (PE) membrane: (a) coiled state in the aqueous phase, (b) folded state in the aqueous phase, and (c) extended state on the membrane surface. (B) mRNA synthesis from the DNA is monitored using two ﬂuorescent oligonucleotide probes that hybridize to the mRNA. On the hybrid formed (inset), the distance between two ﬂuorescence molecules is very close, resulting in FRET. to the PE membrane surface accompanied by the conformational transition was observed only when DNA was encapsulated in a microdroplet of less than ∼60 µ m of diameter, but not in a microdroplet larger than ca. 100 µ m, under a physiological ionic strength condition. The thermodynamic analysis showed that the translational entropy loss of a DNA molecule caused by adsorption, i.e., from being conﬁned in a three-dimensional aqueous phase to being attached to a two-dimensional membrane surface, plays a key role in determining the localization and conformation of DNA under such cell-sized (micrometer space) conﬁnement conditions.18 Over the past decade, the artiﬁcial cell studies, in which the cell-sized phospholipid vesicles encapsulating biochemical molecules are experimentally reconstituted and the molecular reactions and events inside the vesicle are investigated, have been progressing. In these studies, protein synthesis (transcrip- tion and translation), enzymatic reactions and other cellular molecular events are run under cellular environmental conditions (in a micrometer-scale aqueous space surrounded by a phos- pholipid membrane).21-29 One of the purposes and goals of these studies is to examine and elucidate the characteristics and driving forces of the cellular molecular events in a micrometer-scale space, such as the reaction rate and the role of the phospholipid (18) Kato, A.; Shindo, E.; Sakaue, T.; Tsuji, A.; Yoshikawa, K. Biophys. J. 2009, 97, 1678–1686. (19) Pautot, S.; Frisken, B. J.; Weitz, D. A. Langmuir 2003, 19, 2870– 2879. (20) Pautot, S.; Frisken, B. J.; Weitz, D. A. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10718–10721. (21) Noirwaux, V.; Libchaber, A. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17669–17674. (22) Norieaux, V.; Bar-Ziv, R.; Salman, H.; Libchaber, A. Phys. Biol. 2005, 2, 1–8. (23) Pietrini, A. V.; Luisi, P. L. ChemBioChem 2004, 5, 1055–1062. (24) Fiordemondo, D.; Stano, P. ChemBioChem 2007, 8, 1965–1973. (25) de Souza, T. P.; Stano, P.; Luisi, P. L. ChemBioChem 2009, 10, 1056– 1063. (26) Hosoda, K.; Sunami, T.; Kazuta, Y.; Matsuura, H.; Suzuki, H.; Yomo, T. Langmuir 2008, 24, 13540–13548. (27) Urabe, H.; Ichihashi, N.; Matuura, T.; Hosoda, K.; Kazuta, Y.; Kita, H.; Yomo, T. Biochemistry 2010, 49, 1809–1813. (28) Long, M. S.; Cans, A.-S.; Keating, C. D. J. Am. Chem. Soc. 2008, 130, 756–762. (29) Dominak, L. M.; Gundermann, E. L.; Keating, C. D. Langmuir 2009, 26, 5697–5705. membrane as an interface, which are considered to be different from those on a macro-volume scale (test-tube scale). Our observation that the conformational transition of long DNA on the phospholipid membrane, which depends on the conﬁnement volume, is considered to be a distinctive characteristic that appears to emerge under a cell-sized conﬁnement condition. The regulation of gene expression (transcription) is one of the most essential molecular events in living organisms. If we wish to elucidate the relation between these structural transitions of long DNA through interplay with phospholipid membrane and the transcriptional activity of DNA, the transcription reaction should be measured inside individual microdroplets. In a previous study, we developed a method to detect the transcrip- tional activity of a speciﬁc gene on DNA in a microdroplet (Figure 1B).30 The in Vitro transcription reaction in aqueous solution had been monitored using ﬂuorescent oligonucleotide probes, such as the binary ﬂuorescence resonance energy transfer (FRET) probes31 or molecular beacon,32 which hybridize to the speciﬁc mRNA synthesized. In the binary probe method, two ﬂuorescent oligonucleotide probes, where one is labeled with the donor ﬂuorescence molecule (donor probe) and the other with the acceptor ﬂuorescence molecule (acceptor probe), hybridize to an adjacent location on the target mRNA. On the hybrid formed, the distance between two ﬂuorescence molecules becomes very close, which results in FRET. These methods can also be used to visualize a speciﬁc mRNA in living cells under ﬂuorescence microscopy.33-42 We examined the binary FRET (30) Tsuji, A.; Yoshikawa, K. ChemBioChem 2010, 11, 351–357. (31) Sei-Iida, Y.; Koshimoto, H.; Kondo, S.; Tsuji, A. Nucleic Acids Res. 2000, 28, e59. (32) Liu, J.; Feldman, P.; Chung, T. D. Y. Anal. Biochem. 2002, 300, 40– 45. (33) Tsuji, A.; Koshimoto, H.; Sato, Y.; Hirano, M.; Sei-Iida, Y.; Kondo, S.; Ishibashi, K. Biophys. J. 2000, 78, 3260–3274. (34) Ishibashi, K.; Tsuji, A. Anal. Chem. 2003, 75, 2715–2723. (35) Sokol, D.; Zhang, X.; Lu, Ponzy; Gewirtz, A. M. Proc. Natl. Acad. 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