ON-OFF Switching of Long DNA Transcription Figure 3. Confocal microscope images of DOPE microdroplets encapsulat- ing YOYO-1-labeled λZAPII/actin DNA (6 mM Mg2+). (a and d) Transmitted-light images (×20 objective). (b and e) Fluorescence images (×20 objective). (c and f) Fluorescence images (×100 objective). Each dotted circle in (c) and (f) indicates the periphery of a microdroplet. The arrow indicates a DNA molecule, the magniﬁed image of which is shown in the inset. The image acquisition time was 33 ms. Scale bar: 20 µ m. conformational transition of long DNA (more than 20-30 kbp) induced by spermine is of the “all-or-none” (discontinuous) type at the individual DNA molecule level, and all of the DNA molecules have the folded state in the presence of sufﬁciently high concentrations of spermine.18,44 Transcription in Microdroplets. λZAPII/actin DNA (nonla- beled, 0.25 ng/µ L) and ﬂuorescent oligonucleotide probes (4 × 10-8 M, which corresponds to 4000-fold amount (molar ratio) of DNA) were encapsulated in DOPE microdroplets, and these were incubated on the microscope stage. To monitor the transcription inside the microdroplets, ﬂuorescence images in three channels (FRET channel: acceptor ﬂuorescence with donor excitation. Donor channel: donor ﬂuorescence with donor excitation. Acceptor channel: acceptor ﬂuorescence with ac- ceptor excitation) were obtained sequentially at various incuba- tion times. In the absence of spermine, discrete FRET signals diffusing inside the microdroplet appeared in the FRET channel from 10-15 min of incubation (Figure 4a). The intensity of each FRET signal tended to become stronger as incubation progressed from 10 to 30 min, and did not change after 30-60 min. The number of discrete signals remained almost the same after 10-30 min. The similar discrete ﬂuorescence signals appeared in the other two channels, the Donor channel and the Acceptor channel (Figure S2 in the Supporting Information), which indicates that multiple donor and acceptor probes were located in close proximity, giving a discrete ﬂuorescence signal. When the sense probe was added to the reaction solution instead of the antisense probe, such discrete ﬂuorescence signals did not appear in any of the three channels throughout the entire incubation period of more than 3 h (Figure 4b and Figure S2). Also, when incubation was run without RNA polymerase (with the antisense probe), these discrete signals were not observed (44) Takagi, S.; Tsumoto, K.; Yoshikawa, K. J. Chem. Phys. 2001, 114, 6942–6949. A R T I C L E S (images not shown). These results show that the discrete signals were derived from -actin mRNA synthesized inside the microdroplet. In the last study, we performed experiments of transcription from a short plasmid DNA (pBluescript II SK(-) containing -actin cDNA, 5 kbp) in the DOPE microdroplet.30 When a high concentration of DNA was present (5 ng/µ L, which corresponds to ∼30000 transcription sites in a microdroplet with a diameter of 40 µ m), FRET ﬂuorescence was diffusely observed throughout the microdroplet, and the progress of the transcription reaction was monitored by measuring the increase in FRET ﬂuorescence intensity inside the microdroplet,45 as in the case of bulk solution. At a very low DNA concentration, discrete FRET signals, which are similar to those in the present study, were observed inside the microdroplet, and FRET ﬂuorescence in other parts of the microdroplet was within the background level. In this method, each RNA molecule is labeled with a pair of ﬂuorescent probes. Since a single ﬂuorescence molecule dif- fusing in the aqueous solution is not detected as a discrete ﬂuorescence signal under the imaging conditions, the discrete ﬂuorescence signal observed should be considered not to correspond to a single RNA molecule diffusing in the aqueous phase but rather to “multiple RNA molecules”. We conclude that each discrete ﬂuorescence signal represents a “multiple RNAs on a single DNA”, which means a DNA molecule that is being transcribed (more detailed discussion is given in the Footnote S1 in the Supporting Information). Transcriptional Activity Is Inhibited by the Con- formational Transition of DNA to a Folded State. Effects of Spermine. When the spermine concentration increased, the number of discrete ﬂuorescence signals in a microdroplet decreased (Figure 4). The number of discrete FRET signals in the microdroplet images, each of which was obtained at a center confocal plane of the microdroplet, was counted at various spermine concentrations (Figure 5). The number of signals decreased to almost zero at more than 0.6 mM of spermine. We also counted the number of YOYO-1-labeled DNA molecules in the microdroplet images at various spermine concentrations. These values were 14.0 ( 2.32, 12.8 ( 1.46, 13.1 ( 1.68, 12.4 ( 2.44, and 12.7 ( 1.67 at 0, 0.2, 0.4, 0.6, and 0.75 mM of spermine, respectively. These results show that the efﬁcacy of encapsulation of λZAPII/actin DNA into DOPE microdroplets is independent of whether the DNA has a coiled state or a folded state at this very low DNA concentration, and there were almost the same numbers of DNA molecules in the microdroplet at spermine concentrations of 0-0.75 mM. Ac- cording to our conclusion that each discrete ﬂuorescence signal represents a DNA molecule that is being transcribed, almost all of the DNA molecules in the microdroplet were “transcrip- tional-active” in the absence of spermine, while at 0.4 mM of spermine, for example, only one-third of the DNA molecules (45) For λZAPII/actin DNA, ∼30000 transcription sites in a 40 µ m microdroplet correspond to 43 ng/µ L of DNA. It is actually difﬁcult to encapsulate such high concentration of long λZAPII/actin DNA into microdroplets. J. AM. CHEM. SOC. 9 VOL. 132, NO. 35, 2010 12467