A R T I C L E S Tsuji and Yoshikawa Figure 4. Confocal ﬂuorescence microscope images (FRET channel) of DOPE microdroplet (6 mM Mg2+). Representative images of 30-min incubation are shown. The dotted circle and arrows indicate the periphery of a microdroplet and discrete FRET signals inside the microdroplet, respectively. Scale bar: 20 µ m. Figure 5. Number of discrete FRET signals formed by the transcription reaction in DOPE microdroplets with a diameter of 40-60 µ m. For each microdroplet, image acquisition for 0.2 s was repeated three times at a center confocal plane of the microdroplet, and the number of discrete signals in the microdroplet was counted. More than 20 microdroplets were examined at each spermine concentration in two independent experiments. were “transcriptional-active”,46 and none of the DNA was “active” at more than 0.6 mM of spermine.47 We also performed an experiment to examine the effects of spermine on the amount of RNA synthesis. Transcription was run for 30 min in the bulk solution, and synthesized RNA was puriﬁed (Figure 6A). Figure 6B also shows the results when short 2.1 kbp DNA, instead of λZAPII/actin, was used as a template for the transcription reaction. In λZAPII/actin DNA, as the spermine concentration increased, the amount of synthe- sized RNA decreased, and almost no RNA was detected at more than 0.6 mM of spermine. In contrast, with 2.1 kbp DNA, the presence of 0-0.3 mM of spermine did not affect RNA synthesis. When the spermine concentration increased to more than 0.3 mM, RNA synthesis decreased, but some RNA was detected even at 0.75 mM of spermine. (46) As described above, the folding transition of long DNA is a ﬁrst- order phase transition that is discrete at individual DNA molecule level.7,8 Therefore, in the intermediate spermine concentration region, DNA molecules with a coiled conformation and those with a folded conformation coexist. The results and interpretation in Figure 5 are consistent with this fact. (47) The average numbers of discrete FRET signals were not zero, but rather 0.27 and 0.20 at spermine concentrations of 0.6 and 0.75 mM, respectively. At these spermine concentrations, we observed several microdroplets that had only one signal. It is possible that the DNA was cut off to a shorter length during the experimental handling. If the cut-off DNA was too short to exhibit a conformational transition to a folded state, transcription might occur on this DNA (see later discussion on 2.1 kbp DNA). Therefore, we interpret our results to indicate that “none of the (long λZAPII/actin) DNA was “active” at more than 0.6 mM of spermine”. 12468 J. AM. CHEM. SOC. 9 VOL. 132, NO. 35, 2010 Figure 6. Dependence of RNA synthesis on the spermine concentration. The transcription reaction was performed with 1.25 ng/µ L of λZAPII/actin DNA or 2.1 kbp DNA and various concentrations of spermine in 100 µ L of bulk solution (6 mM Mg2+) at 37 °C for 30 min. RNA was puriﬁed from the reaction solution, and the amounts were estimated. In the transcription imaging of λZAPII/actin DNA in the microdroplet, almost all of the DNA molecules were “tran- scriptional-active” at a spermine concentration of 0-0.2 mM (Figure 5). On the other hand, the amount of RNA synthesized in the presence of 0.2 mM of spermine was less (∼60%) than that in the absence of spermine (Figure 6A). These results suggest that fewer RNA molecules were synthesized from a DNA molecule during the incubation period. Since the amount of RNA synthesized from the 2.1 kbp DNA was almost the same under range of spermine concentrations of 0 to 0.2 mM (Figure 6B), the decrease in the amount of RNA synthesis on λZAPII/actin DNA is considered to be not a direct consequence of the effects of spermine on the transcription reaction but rather a consequence of the effects on the structure of long λZAPII/ actin DNA. At 0.2 mM of spermine, λZAPII/actin DNA had a coiled conformation, not a folded conformation. Previously, we noted that the long-axis length of DNA molecules with a coiled conformation observed in the ﬂuorescence images decreased