ON-OFF Switching of Long DNA Transcription in aqueous solution when the concentration of polyamine or Mg2+ increased,18,44 which is caused by the partial neutralization of the negative charges of the phosphate moieties of DNA by Mg2+ or spermine. In these “shrunken” coil structures, the rate of some steps in the transcription reaction, such as the binding of RNA polymerase to DNA, might become lower, which would result in a decrease in the reaction rate of RNA synthesis. The most important result in Figure 6A is that only a very few RNA were detected at 0.5 mM of spermine, and none were detected at more than 0.6 mM of spermine. These results are consistent with those of transcription imaging at individual DNA molecule level, where no discrete FRET signal was observed at more than 0.6 mM of spermine (Figure 5). These results show that all of the DNA molecules with a folded conformation (Figure 3B) are “transcriptional-silent”. On a short 2.1 kbp DNA, RNA synthesis occurred in the presence of more than 0.6 mM of spermine (Figure 6B). When 2.1 kbp DNA was encapsulated in DOPE microdroplets and transcription was run, discrete FRET signals were observed even at 0.75 mM of spermine, although there appeared to be fewer signals than in the absence of spermine (images not shown).48 These results support our conclusion that the complete inhibition of the transcription on long λZAPII/actin DNA in the presence of higher concentrations of spermine was due to the folding conformation of the DNA. With 2.1 kbp DNA, the amount of synthesized RNA decreased when the spermine concentration became higher than 0.4 mM. When we observed ﬂuorescent-labeled 2.1 kbp DNA in the microdroplets, the extensive aggregation and/or condensa- tion of DNA molecules was not observed even at 0.75 mM of spermine. However, the small aggregates of several DNA molecules might be formed,49 on which transcription would be inhibited or suppressed. Another possibility is that high con- centrations of spermine directly affected the reaction rate in some steps of the transcription reaction. Recovery of Transcriptional Activity on an Extended Coil Conformation on Phospholipid Membrane. The λZAPII/actin DNA in a DOPE microdroplet was adsorbed to the membrane inner surface at a high Mg2+ concentration (15 mM, Figure 7A(a-c)). On the membrane surface, the DNA molecule exhibits a quasi-two-dimensional coil state with intramolecular chain motion (movie S1 in the Supporting Information), as in the case of T4 DNA we reported.18 The transcription reaction was run under this condition. From 10-15 min of incubation on the microscope stage, discrete ﬂuorescence signals appeared on the membrane inner surface in all three channels. (An image (48) We encapsulated the same concentration (0.25 ng/µ L) of 2.1 kbp DNA in microroplets as was used for λZAPII/actin DNA; 0.25 ng/µ L of 2.1 kbp DNA corresponds to more than 30000 molecules in a microdroplet with a diameter of 40 µ m. Indeed, there were a large number of discrete FRET signals in the microdroplets. In addition, the diffusion of discrete signals in the aqueous phase was faster than in the case of λZAPII/actin DNA. Therefore, it was actually difﬁcult to count the number of discrete FRET signals in the microdroplets. (49) Under the present imaging conditions, it would be difﬁcult to distinguish between a single 2.1 kbp DNA molecule and an aggregate of several DNA molecules, if it forms. This is due to the much lower signal-to-noise ratio of ﬂuorescence images of a 2.1 kbp DNA diffusing in the aqueous phase. When compared to the long λZAPII/actin DNA, the short 2.1 kbp DNA shows a much faster translational diffusion because it does not exhibit an apparent coiled structural ﬂuctuation. As a result, the residence time of the DNA on focus during an image acquisition (33 ms) becomes very short. Also, the number of ﬂuorescent dyes (YOYO-1) included in a DNA molecule is smaller. These decrease the signal-to-noise ratio of ﬂuorescence images of a 2.1 kbp DNA. A R T I C L E S Figure 7. Confocal microscope images of DOPE microdroplets encapsulat- ing λZAPII/actin DNA (15 mM Mg2+). (A) DNAs labeled with YOYO-1 in a DOPE microdroplet. (a and d) Transmitted-light images (×20 objective). (b and e) Fluorescence images (×20 objective). (c and f) Fluorescence images (×100 objective). In (c) and (f), the arrow indicates a DNA molecule, the magniﬁed image of which is shown in the inset. (B) λZAPII/actin DNA (nonlabeled) and ﬂuorescent oligonucleotide probes were encapsulated in microdroplets, and the transcription reaction was run inside the microdrop- lets. Fluorescence images in the FRET channel at 30 min of incubation (×100 objective). The arrow indicates a discrete signal. Scale bar: 20 µ m. in the FRET channel is shown in Figure 7B(a)). As discussed above, these signals are considered to be derived from multiple RNA molecules on a DNA molecule. These results show that RNA synthesis occurred on the DNA molecule which was located on the membrane surface and had an extended coil conformation. Next, we examined the transcriptional activity of DNA under the coexistence of high concentrations of Mg2+ and spermine. The transcription reaction was run with λZAPII/actin DNA and ﬂuorescent oligonucleotide probes at various concentrations of Mg2+ and spermine in the bulk solution, and the reaction solution was then encapsulated in DOPE microdroplets. Discrete ﬂuorescence signals similar to those observed when transcription was run inside microdroplets were observed. These discrete signals were not observed at high concentrations of spermine. The numbers of discrete FRET signals were counted (Figure 8). The concentration of spermine at which no discrete signal formed increased as the Mg2+ concentration increased. It has been reported that Mg2+ has a competitive effect on the folding transition of long DNA induced by spermine; DNA requires a higher spermine concentration for its conformational transition to the folded state in the presence of a higher concentration of J. AM. CHEM. SOC. 9 VOL. 132, NO. 35, 2010 12469