File S1 - Introducing Micrometer-Sized Artificial Objects into Live Cells: A Method for Cell–Giant Unilamellar Vesicle Electrofusion

Figure S1: Schematic diagram of the square DNA origami structure. M13mp18 ssDNA, its complementary ssDNA sets (called staples), and green fluorescent (FITC)-conjugated oligonucleotides (5′-GCAATGAGTAGATCCTGGCACTCTCGATGCGACAG-3′ and 5′-TGCCAGGATCTACTCATTGC-3′) were purchased from Operon Technologies (Japan) and Takara Bio (Japan), respectively. These DNAs were mixed (M13:staples:FITC  = 4 nM:20 nM:60 nM) and annealed in a buffer (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9, 25°C) for 3.5 h across a temperature range from 95 to 25°C at a rate of -1°C/3 min. Figure S2: Identification of the DNA origami structure. (A) Electrophoresis analysis. Left, middle, and right lanes contain the marker, bare plate-like DNA origami structure, and fluorescently-tagged DNA origami, respectively. The samples were analyzed by 1% agarose gel electrophoresis (100V, 1 hour). The DNA origami structure, with or without fluorescent (FITC) tag, was electrophoresed in a 1% agarose gel that was exposed to DC 100 V for 1 h. FITC fluorescence was detected using a ChemiDoc MP system (BioRad, Japan). A band showing FITC-tagged origami was clearly observed under blue light. (B) AFM images for the DNA origami. The AFM image was obtained on an AFM system (Nano Live Vision, RIBM, Tsukuba, Japan) using a silicon nitride cantilever (resonant frequency  = 1.5 MHz, spring constant  = 0.1 Nm-1, EBDTip radius  = 24 nm, Olympus BL-AC10DS-A2). The sample (2 µL) was adsorbed onto a freshly cleaved mica plate for 5 min at room temperature, and then washed twice with the same buffer solution. Scanning was performed in the same buffer solution using a tapping mode. The final concentration of the DNA (M13mp18) was 100 nM dissolved in buffer (Tris/Tris-HCl 20 mM, Mg2+ 12.5 mM (pH 7.4)). Scale bar  = 100 nm. Figure S3: Size distribution of the formed GUVs. To confirm the size distribution of the GUVs, we prepared GUVs with the inner buffer of 40 µM Lucifer yellow (SIGMA, Japan), 300 mM mannitol, 0.1 mM CaCl2, 0.1 mM MgCl2. (A) Representative image of Lucifer yellow contained GUVs. Scale bar  = 50 µm. The fluorescent microscopic images of GUVs obtained immediately after water-in-oil emulsion transfer method. (B) The binarized image from (A). The size distribution was calculated from the images by using Image J software (NIH). (C) Size distribution of the Lucifer yellow contained GUVs diameter (n = 471). The average diameter is 37±13 µm, as mean ± standard deviation. Figure S4: Representative “Pearl-chain” form of GUVs and HeLa cells suspension. Each vesicle contained fluorescent beads of a particular size, e.g., 200 nm, 500 nm, 1 µm, and 2 µm in diameter. The coupled micrographs of phase contrast and fluorescence image show the same position of the sample immediately before the electrofusion. Scale bar  = 50 µm. Figure S5: Confocal microscopic images of HeLa cells containing the introduced fluorescent beads. (A) Microscopic images showing the cross-section of the treated HeLa cells. These images show HeLa cells into which (from the top) no beads, or beads of 0.2 µm, 0.5 µm, 1 µm, or 2 µm diameter (green) had been introduced. Z-stack acquisitions were performed to detect the position of the beads from the dorsal (left column) to the ventral (right column) cross-section of the cell. Cells were stained with cell tracker and Hoechst to reveal the cytoplasm (red) and nucleus (blue). Scale bar  = 10 µm. (B) 3-D reconstruction of images of HeLa cells containing 1-µm microbeads, obtained from confocal microscopic image stacks of a birds-eye view (upper left), side view (lower left), and top view (right). Sky blue, white line, and green dotted line indicate the bottom of the cells, slice position of the top view, and side view, respectively. Table S1: Sequences for the plate-like DNA origami structure.
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