A R T I C L E S Figure 2. (A (a)) λZAPII DNA containing -actin cDNA. -actin cDNA with the terminator sequence of T7 RNA polymerase with the 3′-end was inserted into the multicloning site of λZAPII DNA.(A (b)) PCR fragment DNA from λZAPII/actin DNA. (B) Pair of ﬂuorescent oligonucleotide probes that hybridize to a site of -actin mRNA. (C) Fluorescence spectral changes with the progression of the transcription reaction from λZAPII/ actin DNA (for details, see the Methods in the Supporting Information). probe method to monitor a transcription reaction in a micro- droplet. In the study, a plasmid DNA, ﬂuorescent oligonucleotide probes for the mRNA, and other transcriptional reaction components were encapsulated in microdroplets, and the progress of the transcription reaction inside individual micro- droplets could be monitored by measuring FRET ﬂuorescence intensity under ﬂuorescence microscopy.30 In this study, to elucidate the relation between the DNA conformational transition and its function in a cell-sized microdroplet coated with phospholipid membrane, we prepared a long DNA containing a gene transcribed under in Vitro conditions (λZAPII DNA containing human -actin cDNA, 43 kbp, Figure 2A). The DNA was encapsulated in microdroplets, in which the location and conformational state of DNA and transcriptional activity were investigated under ﬂuorescence microscopy. The ON or OFF of transcriptional activity was evaluated at individual DNA molecule level, which showed that transcription is completely inhibited in the folded DNA molecule in the aqueous phase while it is reactivated when the DNA (39) Tyagi, S.; Alsmadi, O. Biophys. J. 2004, 87, 4153–4162. (40) Dirks, R. W.; Molenaar, C.; Tanke, H. J. Methods 2009, 6, 331–338. (41) Bao, G.; Rhee, W. J.; Tsourkas, A. Annu. ReV. Biomed. Eng. 2009, 11, 25–47. (42) Tyagi, S. Nat. Methods 2009, 6, 331–338. 12466 J. AM. CHEM. SOC. 9 VOL. 132, NO. 35, 2010 Tsuji and Yoshikawa interacts with phospholipid membrane in the presence of higher concentrations of Mg2+. Results Experimental System. We constructed the λZAPII vector DNA containing human -actin cDNA (λZAPII/actin DNA, Figure 2A(a)). On the DNA, a single promoter sequence of T7 RNA polymerase is located upstream of the -actin cDNA sequence. This DNA is ∼43 kbp in length and exhibits a conformational transition between a coiled state and a folded state in aqueous solution (described later). To examine the effects of DNA length in some experiments, we prepared a short (2.1 kbp) DNA fragment that contains the T7 promoter and -actin cDNA sequence (Figure 2A(b)). We also prepared a pair of ﬂuorescent oligonucleotide probes for the 386-425 site of -actin mRNA (antisense probe, Figure 2B). These are 19-mer 2′-O-methyl RNA, each of which is labeled with AlexaFl488 (donor) at the 3′-end or with Cy5 (acceptor) at the 5′-end.30 These two probes hybridize to -actin mRNA adjacent to each other, resulting in FRET.30,33 We also prepared a pair of ﬂuorescent oligonucleotide probes (that have the same sequence as the 386-425 site of -actin mRNA), which do not hybridize to -actin mRNA (sense probe).30 Figure 2C shows the ﬂuorescence spectral changes of the reaction solution when transcription was carried out in the bulk aqueous solution with λZAPII/actin DNA and 300-fold amounts (molar ratio to DNA) of antisense ﬂuorescent oligonucleotide probes. As in the case of a plasmid DNA containing -actin cDNA,30 donor ﬂuorescence intensity (520 nm) decreased, and acceptor ﬂuorescence intensity (670 nm) increased as incubation progressed, which indicates that the FRET efﬁciency increased with time. This result shows that -actin mRNA synthesis occurred from λZAPII/actin DNA, which can be monitored by measuring FRET. When the transcription reaction was carried out with the 2.1 kbp DNA fragment, similar ﬂuorescence spectral changes were observed (data not shown), which show that the 2.1 kbp DNA fragment also acts as a template for the transcription of -actin mRNA. DNA in Microdroplets. λZAPII/actin DNA molecules were labeled with ﬂuorescent dye, YOYO-1, and those in the transcription buffer (6 mM Mg2+) containing various concentra- tions of spermine were encapsulated in water-in-oil microdrop- lets composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanola- mine (DOPE). Figure 3 shows confocal microscope images of the microdroplets. At this Mg2+ concentration, DNAs were located in the aqueous phase. At a very low DNA concentration (0.25 ng/µ L, which corresponds to less than 200 DNA molecules in a microdroplet with a diameter of 40 µ m), each DNA molecule diffusing inside the droplet can be observed (Figure 3, c and f). In the absence of spermine (c), DNA had a coiled conformation, while in the presence of 0.6 mM spermine it had a highly compact (folded) conformation (f), as the case of T4 DNA we previously reported.18,43 These results show that λZAPII/actin DNA exhibits a conformational transition between a coiled state and a highly folded state, depending on the concentration of spermine. Our previous studies show that the (43) The concentrations of Mg2+ and spermine at which λZAPII/actin DNA exhibits a conformational transition from a coiled state to a folded state are different from those for T4 DNA,18 due to differences in DNA length (T4 DNA; 166 kbp, λZAPII/actin DNA; 43 kbp) and the ionic strength of the solution. The composition and concentrations of monovalent ions used in this study are those for the optimal activity of T7 RNA polymerase in Vitro.