Published on Web 08/12/2010 ON-OFF Switching of Transcriptional Activity of Large DNA through a Conformational Transition in Cooperation with Phospholipid Membrane Akihiko Tsuji*,† and Kenichi Yoshikawa*,†,‡ Spatio-Temporal Order, ICORP, Japan Science and Technology Agency, Tokyo 102-0075, Japan, and Department of Physics, Graduate School of Science, Kyoto UniVersity, Kyoto 606-8502, Japan Received June 13, 2010; E-mail: tsuji@chem.scphys.kyoto-u.ac.jp; yoshikaw@scphys.kyoto-u.ac.jp Abstract: 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, 97, 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. Introduction Long DNA molecules larger than 20-30 kilo base-pairs (kbp) have the characteristics of a semiﬂexible polymer and exhibit a large discrete conformational transition from a coiled state to a highly folded state in aqueous solution, in the presence of various condensing agents including cellular environmental factors such as polyamine.1-6 This transition is discrete at an individual DNA molecule level and can be described in terms of a ﬁrst-order phase transition under the criterion of Landau.7,8 In the folded conformation, it has been reported that the biologically important function of DNA, transcription, is completely inhibited.9,10 † Japan Science and Technology Agency. ‡ Kyoto University. (1) Bloomﬁeld, V. A. Biopolymers 1991, 31, 1471–1481. (2) Bloomﬁeld, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334–341. (3) Marquet, R.; Wyart, A.; Houssier, C. Biochim. Biophys. Acta 1987, 909, 165–172. (4) Marquet, R.; Houssier, C. J. Biomol. Struct. Dyn. 1991, 9, 159–167. 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SOC. 2010, 132, 12464–12471 The interaction of DNA with zwitterionic phospholipid such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the presence of divalent metal cations has been extensively investigated.11-17 Recently, we found the conformational transi- tion of DNA through interplay with phospholipid membrane, from a folded state in the aqueous phase to an extended coil state on the phospholipid membrane, in a cell-sized microdroplet (Figure 1A).18 In a phospholipid-coated water-in-oil micro- droplet19-21 that encapsulated bacteriophage T4 DNA (166 kbp), the DNA was located in the aqueous phase with a folded conformation in the presence of a tetravalent amine, spermine (b). At a high Mg2+ concentration, DNA was adsorbed onto the membrane surface and exhibited an extended conformation when the membrane was composed of PE (c), but not when it was composed of PC. More interestingly, the adsorption of DNA (11) Lis, L. J.; Lis, W. T.; Paresgian, V. A.; Rand, R. P. 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