A Fissionable Artificial Eukaryote-like Cell Model - Journal of the

Jul 5, 2017 - The use of artificial cells has attracted considerable attention in various fields from biotechnology to medicine. Here, we develop a ce...
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A Fissionable Artificial Eukaryote-like Cell Model Wei Zong,† Shenghua Ma,† Xunan Zhang, Xuejing Wang, Qingchuan Li, and Xiaojun Han* State Key Laboratory of Urban Water Resource and Environment, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, 92 West Da-Zhi Street, Harbin 150001, China S Supporting Information *

ABSTRACT: The use of artificial cells has attracted considerable attention in various fields from biotechnology to medicine. Here, we develop a cell-sized vesicle-in-vesicle (VIV) structure containing a separate inner vesicle (IV) that can be loaded with DNA. We use polymerase chain reaction (PCR) to successfully amplify the amount of DNA confined to the IV. Subsequent osmotic stress-induced fission of a mother VIV into two daughter VIVs successfully divides the IV content while keeping it confined to the IV of the daughter VIVs. The fission rate was estimated to be ∼20% quantified by fluorescence microscope. Our VIV structure represents a step forward toward construction of an advanced, fissionable cell model.

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INTRODUCTION Cells constitute the basic structural and functional unit of all known organisms. Their foundational nature is emphasized by the fact that cells are the smallest “living” entity of every organism.1 Cells effectively represent the ultimate model for the development of life, and a considerable amount of research2 aims at developing equivalent synthetic systems-artificial cells. The development of artificial cells could contribute to the increased understanding of the fundamental processes in natural cells, and provide opportunities for new applications of such synthetic systems.3 It is generally accepted that three basic elements are needed for the construction of a “living” artificial cell: holding information-carrying molecules, compartmentation, and sustaining a metabolic system.4 Additionally, the reproduction of artificial cells is considered as another paramount element for understanding the survival of life.5 Currently, the state-of-the-art artificial cells are “vesicle-invesicle (VIV)” structures comprising subcompartments to mimic the hierarchical architecture of eukaryotic cells.5,6 The reported VIV structures included liposome-in-capsule,7 polymersome-in-polymersome,8 and liposome-in-liposome architectures.9 The common formation methods were layer-bylayer assembly,7 hydration,10 microinjection,11 microfluidic method,12 and shape transformation from unilamellar vesicles upon external stimuli (pH,13 temperature,14 or osmotic stress15). The main problem for VIV structures to mimic eukaryotic cells was the complicated procedures for the preparation of VIV structures, and the deficient molecular loading, which caused difficulty for the development of complex reactions in the inner vesicles, such as DNA amplification. Moreover, the complicated VIV structures also led to an increased difficulty in creating a successful reproduction mimicking the division behavior of eukaryotic cells. The cell © 2017 American Chemical Society

division currently was mainly mimicked using single compartment unilamellar vesicles.1b,16 Osmotic stress has been extensively used to induce the shape deformation of unilamellar vesicles.16c,17 It causes a water volume change inside the vesicles, variation of specific surface area, and subsequent shape transformation.18 Although the endocytosis and exocytosis of vesicles have been reported for giant unilamellar vesicles (GUVs) upon osmotic stress, quite diverse unintended shape deformations including starfish-like structures, tubes, or multilamellar vesicles were constantly observed.19 A controlled molecular loading and shape deformation for GUVs was seldom reported from osmotic stress. Herein, we demonstrated the formation of VIV lipid structures by controlled internalization of certain number of vesicles (only one vesicle inside a vesicle in our case) and molecular loading of biomacromolecules inside the inner vesicle (IV) using osmotic stress, with the capability of amplifying DNA via polymerase chain reaction (PCR) inside a separated IV. Our VIV structure can also undergo osmotic stress induced fission into two daughter VIVs, each containing an IV with material from that of the mother VIV. Our VIV structure presents an important step forward toward the development of synthetic living cells.

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RESULTS AND DISCUSSION The results are presented following the chronology of the experiment, with first the preparation of the VIV structures from GUVs, then the subsequent loading of the IV with DNA and PCR materials, followed by DNA amplification. Finally, we Received: April 20, 2017 Published: July 5, 2017 9955

DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960

Article

Journal of the American Chemical Society

believed to mainly involve two factors: molar flux of water (J) through the vesicle membrane and membrane rigidity. With steady water loss, the vesicle tends to form VIV structures. At 25 °C, both DMPC and POPC membranes are fluid. The suitable JDMPC leads to a steady water loss, consequently to form VIVs. The higher JPOPC causes POPC vesicles to undergo dramatic fluctuations to form a starfish shape. Although JDPPC is small, the rigidity of DPPC membrane at 25 °C causes a slower response to same osmotic shock, and to maintain a nonequilibrium state for a certain period of time. These results confer that our DMPC vesicle possesses ideal properties for a VIV structure, with the IV mimicking a cell “nucleus” and the OV lipid bilayer the “cell membrane”. To obtain more stable VIV structures, DMPC GUVs containing 30%, 50%, and 70% cholesterol (mol %) were fabricated in water solution and allowed to deform triggered by osmotic stress from 12.5 mM (ΔΠ = −0.31 atm) glucose solution (Figure 1d−f). Cholesterol is a ubiquitous component in cell membrane, where it plays an important stabilizing role.24 Here, we could clearly observe that DMPC GUVs containing 30 mol % cholesterol offer the best results for VIV structures (Figure 1d). This is consistent with the fact that the concentration of cholesterol is always in the range between 30 and 40 mol % of plasma membrane lipids, despite variations in lipid compositions among different types of cells.25 Hereafter, we systematically use 30 mol % cholesterol in our VIV structures. The inset of Figure 1d was obtained with the same conditions as in Figure 1d, but with 0.01 mg mL−1 rhodamine added to the triggering solution so as to evidence trapping of the triggering solution inside the IV. The rhodamine molecules outside OV were removed by dialysis. Significantly, our method allows us to load the IV and the OV with any material of our choosing. To demonstrate the possibilities offered by our VIV structure, we proceeded to load the IV with material suitable for DNA PCR amplification. First, GUVs were produced in PCR buffer solution. Next, DNA templates and PCR components (including polymerase, primers and deoxyribonucleoside triphosphates (dNTPs), MgSO4, and Green qPCR SuperMix) were added into the GUV solution aimed to cause the hypertonic shock. This resulted in only the IV being loaded with DNA and other PCR components, while the OV contained only PCR buffer (Figure S2). The successful formation rate of the VIV structure was 78.2 ± 2.7% (n = 200) as assessed by analyzing the events in microscope images. The morphology of GUVs and their deformation upon osmotic stress can be described with the area difference elasticity (ADE) theory,26 which models the energy as a sum of two terms: one dependent on curvature at any point on the surface, and the second dependent on the curvature-induced area difference between the inner and the outer leaflets of the lipid bilayer. The ADE theory predicts that the reduced volume (v), defined as the ratio v = 3(4π)1/2V/A3/2 between the inner volume enclosed by the membrane (V) and the volume of a sphere with the same area (A), is the critical parameter determining the vesicle shape (Figure 2a). Sphere vesicles evolve to stomatocytes once v is close to 0.59 (reducing from larger values, Figure 2b), and evolve to the VIV structure observed here as v further reduces (Figure 2c). According to the above-mentioned formula, the experimental value of v for the VIVs is vexp = 0.58 ± 0.05 (n = 300). The vexp matches the theoretical prediction, which confirms the validity of ADE theory to explain the formation of VIV structures. Exper-

show how the VIV can be divided into two intact daughter VIVs. Every result shown in this Article was repeated at least five times. Preparation of the VIVs. The simplest strategy to affect the shape of GUVs is through hypertonic shocks.20 The osmotic pressure is calculated from the concentration difference between inner and outer vesicle media using the following equation: ΔΠ = RT(cint − cext) = RTΔc. In practice, the response of GUVs to osmotic stresses depends on lipid composition, and we explored the effect of osmotic stress on GUVs composed of POPC, DMPC, and DPPC, examining in particular their shape deformation. POPC, DMPC, and DPPC GUVs were chosen because of their different mechanical properties at room temperature (25 °C). The phase transition temperatures (Tm)21 of POPC, DMPC, and DPPC are −2, 24, and 41 °C, respectively, ensuring different mobility and surface rigidities at 25 °C. The GUVs were initially produced in pure water by electroformation.22 A glucose solution was mixed with the GUV solution for 10 min to induce some deformation. The use of glucose for inducing osmotic stress is well established in the literature.15,23 A homemade setup was used to observe the vesicle morphology under a fluorescence microscope (Figure S1). Microscope images of the GUVs deformation are shown in Figure 1a−c. POPC GUVs tend to adopt a “starfish” shape

Figure 1. Deformation of vesicles (pure water inside) composed of (a) POPC, (b) DMPC, and (c) DPPC under hypertonic conditions triggered by glucose solution (12.5 mM, ΔΠ = −0.31 atm). The deformation of DMPC GUVs containing different cholesterol proportions under hypertonic conditions: (d) 30 mol %, (e) 50 mol %, and (f) 70 mol %. VIV structure of the inset in (d) was obtained under conditions similar to those in (d) but with rhodamine (red fluorescence) inside the IV. The lipid bilayer was labeled with 1 mol % NBD-PE (green fluorescence). The scale bars are (a−c) 10 μm and (d−f) 20 μm.

(Figure 1a), while DMPC GUVs form VIV structures (Figure 1b). Although the osmotic stress is identical in each case, DPPC GUVs shrink upon addition of glucose solution (Figure 1c). The driving force behind the formation of the VIVs is the difference in osmotic pressure inside and outside of GUVs that induces first deformation and subsequently the formation of a separate inner vesicle. To balance the high osmotic pressure of the outside solution, water effectively moves out of the membrane of the outer vesicle (OV). This mechanism allows balancing the osmotic pressure and prevents bursting of the vesicles. The osmotic pressure-induced deformation of GUVs is 9956

DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960

Article

Journal of the American Chemical Society

suitable mixture of DNA and PCR components, we proceeded to demonstrate DNA amplification directly inside the IV. DNA Amplification Inside the IV. The forward primer (5′-TCCAGTGTGGTGGAATTGCCCTT-3′) and the reverse primer (5′-TGCTGGATATCTGCAGAATTGCCCT-3′) were used to amplify PPP1R13L gene (Supporting Information S4) within the IV using the classical PCR technique. To exclude the possibility of undesirable DNA oligomers being amplified outside the VIV structure, the DNA template remaining in the exterior aqueous phase was removed via DNase I digestion. DNase I only digests exposed DNA and cannot traverse the IV membrane to react with DNA located inside (Figure S4). The frequent temperature changes in the PCR process tend to cause strong convection currents, which result in significant shearing forces on GUVs. It is therefore necessary to carry out a test of the VIV structure’s robustness. We tested the VIVs thermal stability for 5, 10, 15, 20, 25, 30, and 35 thermal cycles (Figure S5). The morphology of the VIVs remained intact even after 25 thermal cycles, but progressively adopted an oligovesicular shape after 30 thermal cycles. This is consistent with results reported for simple lipid vesicles in similar thermal conditions.16a,27 Practically, we used 25 thermal cycles for the DNA amplification to ensure the stability of our VIV structures. The success of the amplification process was evaluated by agarose gel electrophoresis analysis of solution extracted from the IVs. To ensure that only DNA products from the IVs were analyzed, DNase I was used to digest any DNA template located outside the IV before and after thermal treatment cycling. The effectiveness of the DNase I was tested by adding DNase I (0.04 unit μL−1) to a conventional PCR solution that had undergone 25 thermal cycles. The resulting solution was kept at 37 °C for 30 min. This resulted in the DNase I degrading all of the amplified DNA molecules with no visible

Figure 2. Schematic illustration of the transformation pathway for a spherical vesicle (a) to a stomatocyte shape (b), to the VIV structure (c) upon osmotic stress. The light blue and dark blue colors represent the initial internal and external solutions of the GUV, respectively. Experimental fluorescence images of stomatocyte (d) and VIV structure (e) formed via GUVs containing 94 mM PCR buffer solution inside triggered by 132 mM (ΔΠ = −0.93 atm) PCR solution outside. The lipid bilayer was labeled with 1 mol % NBD-PE (green fluorescence). The scale bar is 10 μm.

imentally the dynamics of the evolution from GUV to stomatocyte shape is rather fast, at the limit of our observation capabilities with optical microscope. First, the GUV begins to invaginate from a small pore, and the neck of the stomatocyte gradually narrows to a transition state (Figure 2d), which then turns into a VIV structure (Figure 2e) after 30 ± 8 s (n = 200). The formation process of the VIV structures is shown in the supplementary movie 1 and Figure S3. The force driving the shape transformation is the osmotic pressure between the solution outside and inside the GUVs. Once loaded with a

Figure 3. (a) Agarose gel image of amplified DNA oligomers using PCR technique in GUV free solution without (column 1) and with (column 2) DNase I treatment; and in the VIV structures without (column 3) and with (column 4) DNaseI treatment. (b) Agarose gel image of amplified DNA oligomers in the VIV structures after 5, 10, 15, 20, and 25 thermal cycles. (c) Corresponding intensity of the bands in image (b) as a function of thermal cycles. (d) Fluorescence images of amplified DNA oligomers in VIV structure after 25 cycles: membrane image (left), IV image (middle), and merged image (right). The lipid bilayer was labeled with 1 mol % Texas Red DHPE (red fluorescence), while DNA molecules were labeled with SYBR Green I (green fluorescence). The scale bar is 20 μm. 9957

DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960

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Journal of the American Chemical Society band in the gel electrophoresis (Figure 3a, column 2 of the gel). By contrast, a bright DNA band is visible for electrophoresis conducted on the same PCR sample, but without DNase I treatment (Figure 3a, column 1). When the DNA molecules were amplified inside the IVs, a clear bright band was observed in the gel electrophoresis (column 4 in Figure 3a). The band is slightly thinner than that formed from the VIV structure without DNase I treatment (column 3 in Figure 3a). The size of the DNA oligomers produced is 1.1 kb. The quantity of DNA oligomers amplified inside the IVs was investigated as a function of number of thermal cycles, and the results are shown in Figure 3b for 5, 10, 15, 20, and 25 thermal cycles. For less than 10 cycles, little DNA can be detected. After 10 cycles, the amount of oligomers increases with each cycle (Figure 3b and c). The fluorescence intensity comes from the double-stranded DNA-SYBR Green I complex (dsDNA-SG complex).28 To evaluate the PCR amplification efficiency inside the inner vesicles of VIVs (Ev), a real time PCR system was used to obtain Ev of 0.95 (R2 = 0.99); meanwhile, the standard amplification efficiency in PCR solution (Es) was obtained to be 1.02 (R2 = 0.98). These values confirm that the efficient PCR happened inside inner vesicles of VIVs. Fluorescence images of the VIV structures after 25 cycles are shown in Figure 3d. The lipid bilayers contain 1 mol % Texas Red DHPE to highlight the inner and outer vesicles, and the amplified DNA appears green. The merged image (Figure 3d, right) clearly demonstrates that the amplification of DNA occurred selectively, only inside IV. This also demonstrates control of the function of the IV. Following the successful DNA amplification in the IV, we tested the possibility of VIV structure fission in a process mimicking “mitosis”. Fission of the VIV Structures. Division is one of the fundamental properties of a protocell by analogy to “reproduction”. Here, we succeeded in artificially inducing the spontaneous fission of VIV structures without significant loss of IV material or contamination from the “cytoplasm”. Figure 4a illustrates the process for a DNA-amplified VIV structure. When a VIV containing one IV is triggered by osmotic stress, both the OV and the IV are stretched and narrowed in the

center regions, creating a small concentric neck (Figure 4a, step 1, and Figure S6). Subsequently, the VIV undergoes a fission process resulting in two daughter VIVs, each containing a DNA-loaded IV (Figure 4a (step 2)). The full fission process could be continually observed under a fluorescence microscope for a DNA-amplified VIV structure (Figure 4b). The yellowgreen color indicates the presence of a large amount of amplified DNA oligomers inside the IV. The fission is first triggered by the osmotic pressure caused by 23 mM concentration difference (ΔΠ = −0.56 atm) between the solutions outside and inside VIV. The OV is then elongated to an oval shape, together with IV (Figure 4b1). A further increase of the osmotic pressure (81 mM, ΔΠ = −1.99 atm) is obtained by adding a highly concentrated PCR buffer solution, which induces the elongated VIV to form a narrow neck between two communicating proto-VIVs after 158 s (Figure 4b2). The VIV structure eventually completes its fission 46 s later, creating two distinct daughter VIVs (Figure 4b3). The elongation of VIV induced by the first osmotic pressure is believed to be more of a thermodynamic process, while the following fission process triggered by the second osmotic pressure is more of a kinetic process. A full fission process is shown in supplementary movie 2. Statistical analysis of the total transition time (from the stage in Figure 4a1 to Figure 4a3) yielded 213 ± 19 s (n = 100). The VIV fission rate was estimated to be ∼20% (n = 100) by counting VIVs in microscope images. These results indicate that the VIV structure is a fissionable cell model.

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CONCLUSIONS We create VIV lipidic structures by forcing a controlled deformation and reshaping of GUVs using osmotic stress. The high concentration of salt outside the GUVs reduces the volume of the inner compartment, and consequently makes it deform. The formation of the VIV structures is quantitatively explained by the ADE model. Although VIV structures have previously been produced using the hydration of lipids, our method exhibits a significant advantage: the content of the IV can differ from that of the surrounding OV environment, here containing DNA and PCR agents. With DNA carrying the genetic information inside the IV, the VIV structure becomes a model “eukaryocyte”. The classical PCR technique could be successfully carried out to amplify DNA molecules exclusively inside the IV structure, confirmed by fluorescence microscopy and agarose gel electrophoresis. We could observe that DNA with a size of 1.1 kb was produced from a DNA template in the IV. The overall stability of the VIV structure is remarkable with the loaded IV remaining intact for more than 2 weeks, even after thermal cycling during the PCR process. Cell division is a fundamental process to life, with most artificial divisions accomplished with single compartment GUVs. The fission of VIV structure is hence challenging. With osmotic stress triggering, a mother VIV structure could be successfully split into two daughter VIVs, each containing an IV with material from that of the mother VIV. In summary, the VIV structure can reproduce key characteristics of live biological cells such as the presence of an IV that locally holds some information material (DNA molecules), which could be amplified in situ by PCR. Our novel VIV structure can also be divided into two daughter VIVs. The results suggest that osmotic stress might have aided the division of primitive cells prior to the onset of internally regulated and energy-dependent processes used by all cells today.

Figure 4. (a) Schematic illustration of the fission process of a DNAamplified VIV. (b) Fluorescence images of a single VIV at rest (1), elongated (2) stage, and the resulting two daughter VIVs containing amplified DNA molecules inside their respective IV (3). The lipid bilayer was labeled with 1 mol % Texas Red DHPE (red fluorescence), and DNA molecules were labeled with SYBR Green I (green fluorescence). The scale bar in (b) is 20 μm. 9958

DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960

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of PCR products were carried out on a Gel Imaging System (Tanon 1600R, China). An ABI 7300 real time PCR system (Applied Biosystems, U.S.) was used to determine amplification efficiencies. Detection of Amplified DNA. PCR products in the VIV structure were retrieved by the following procedure. Tris-saturated phenol solution was added to the above-mentioned solution after DNase I treatment with volume ratio of 1:1 to make an emulsion. The mixed solution was then centrifuged for 10 min with a centrifuge (Thermo Fisher ST 16/ST16R, U.S.) at 8000g and 4 °C. The supernatant was collected and mixed with equivoluminal chloroform/iso-amyl alcohol (24/1, v/v). After the consequent mixture was centrifuged for 10 min with 8000g at 4 °C, the supernatant was collected and mixed with sodium acetate (3 mol L−1, 1/10 of total supernatant volume) and cold ethanol (2 times the total supernatant volume). Finally, the DNA molecules were obtained after centrifuging followed by discarding the supernatant. To purify DNA, 1 mL of 70% ethanol was added into the tube with shaking. The solution was centrifuged for 5 min at 7500g at 4 °C. The supernatant was removed, and then the sediment species were dried. Before the purified DNA oligomers were analyzed by agarose gel (1.0%) electrophoresis, 100 μL of TE (Tris-HCl/EDTA) buffer solution was added into the sample tube to dissolve DNA. Fission of DNA-Amplified VIV Structure. The concentration of balanced DNA-amplified VIV solution is 174 mM, which was mixed with PCR buffer solution (220 mM, 1/1, v/v) to obtain an overall concentration of 197 mM for 8 min at room temperature. Once VIV was prolonged, the solution was mixed with 439 mM PCR buffer solution (2/1, v/v) to obtain 278 mM solution outside OV to induce VIV fission. The VIV membrane was labeled with 1 mol % Texas Red DHPE (red fluorescence).

MATERIALS AND METHODS

Materials. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol (Chol) were purchased from Avanti Polar Lipids (U.S.). Fluorescence-labeled 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE) and 1,2-dihexadecanol-snglycero-3-phosphethanolamine, triethylammonium salt (Texas Red DHPE) were obtained from Molecular Probes (Eugene, OR). The DNA templates (inhibitory member of the ASPP family) were obtained from Invitrogen Corp. (U.S.). DNA primers were purchased from Comate Bioscience Co. Ltd. (China). EasyTaq DNA polymerase, DNase I, high pure dNTPs, and SYBR Green I dye were bought from TransGen Biotech (China). Tris-saturated phenol was purchased from HaoYang biological reagents (China). Chloroform, iso-amyl alcohol, ethanol, sodium acetate, ethylenediaminetetraacetic acid (EDTA), tris(hydroxymethyl)aminomethane, rhodamine, and hydrochloric acid were obtained from Sigma (China). Biowest Agarose was purchased from Gene Co. (Spain). Glucose was purchased from XiLong Chemical Co., Ltd. (China). Glass slides coated with indium tin oxide (ITO, sheet resistance ≈ 8−12 Ω, thickness ≈ 160 nm) were purchased from Hangzhou Yuhong Technology Co. Ltd. (China). Preparation of GUVs. The GUV was fabricated using electroformation method.29 ITO-coated glass coverslips (25 mm × 45 mm) were cleaned in ethanol and water each for 15 min by sonication and then dried by N2. Lipid of POPC, DMPC, or DPPC including additional fluorescent lipid was dissolved in chloroform to make a solution concentration of 5 mg mL−1. The lipid solution (7.5 μL) was deposited on ITO electrode surface using a needle to spread carefully back and forth six times, followed by drying under vacuum for 2 h. The coverslips were separated by a rectangular polytetrafluoroethylene (PTFE) spacer with a length, width, and height of 35, 25, and 2 mm, respectively. The AC-electric field (5 V, 10 Hz) was applied for 4 h to generate pure water containing GUVs, while AC-electric field (5 V, 100 Hz) was applied for 10× (diluted 10 times of stock solution) PCR buffer solution containing GUVs. Preparation of VIV Structure. Lipid solution (5 mg mL−1) was composed of cholesterol, DMPC, and NBD-PE (or Texas Red DHPE) at a 30:69:1 molar ratio. GUVs containing PCR buffer solution (10×) were produced as mentioned above. The PCR solution (500 μL) and GUV solution (500 μL) (1/1, v/v) were mixed together for 30 min at room temperature to obtain VIV structure via osmotic stress. Here, the PCR solution is different with PCR buffer solution. The PCR solution (500 μL) contained deionized water (82 μL), PCR buffer solution (10×, 50 μL), dNTP mix (2.5 mM/each, 40 μL), DNA templates (40 nM, 10 μL), forward primers and reverse primers (10 μM, 10 μL, each), EasyTaq DNA polymerase (5 U μL−1, 8 μL), MgSO4 (50 mM, 30 μL), TOP Green qPCR SuperMix (2×, 250 μL) and Passive Reference Dye (50×, 10 μL). The dye molecules were added in the solution to visualize amplified DNA. The morphology of VIV was studied with a fluorescence microscope (Nikon 80i, Japan). The radius of vesicle was measured using its embedded NIS elements software. PCR in the VIV Structure. Amplification of DNA was done by a PCR instrument (Thermo Fisher Applied BiosystemsGeneAmp9700 PCR). Before conducting PCR inside IV, excessive DNase I was added to above-mentioned VIV solution to digest the DNA templates and primers in the exterior of OV for 30 min at 37 °C, as illustrated in Figure S2d. The VIV solution was then treated with a thermal cycler under the following thermal conditions: 94 °C for 2 min, [94 °C for 30 s, 60 °C for 30 s, and 72 °C for 70 s] × 25 cycles. After the thermal cycles, the sample was slowly cooled to 4 °C. Finally, excessive DNase I was added again to the solution of VIV (at room temperature, for 30 min) to digest DNA fragments, which might be leaked out from the VIV structure (Figure S2e). After DNase I treatment, the solution of VIV dispersion was heated at 80 °C for 10 min to inactivate DNase I.30 Separation and purification of DNA were completed by centrifugation (Thermo Fisher ST 16/ST16R, U.S.). Consequently, the amplified DNA was analyzed using an electrophoresis analyzer (Beijing Liuyi Instrument Plant DYY-6C, China). Agarose gel images

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b04009. Experimental section and figures (PDF) Real-time formation of VIV (AVI) Real-time fission of VIV (AVI)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Xiaojun Han: 0000-0001-8571-6187 Author Contributions †

W.Z. and S.H.M. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant nos. 21528501 and 21273059). REFERENCES

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DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960

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DOI: 10.1021/jacs.7b04009 J. Am. Chem. Soc. 2017, 139, 9955−9960