Defined DNA-Mediated Assemblies of Gene-Expressing Giant

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Defined DNA-Mediated Assemblies of Gene-Expressing Giant Unilamellar Vesicles Maik Hadorn,†,∥ Eva Boenzli,*,†,∥ Kristian T. Sørensen,† Davide De Lucrezia,‡ Martin M. Hanczyc,† and Tetsuya Yomo*,§ †

Center for Fundamental Living Technology (FLinT), Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark ‡ Explora S.r.l., Rome, Italy § Graduate School of Information Science and Technology, Osaka University, Osaka, Japan ABSTRACT: The technological aspects of artificial vesicles as prominent cell mimics are evolving toward higher-order assemblies of functional vesicles with tissuelike architectures. Here, we demonstrate the spatially controlled DNA-directed bottom-up synthesis of complex microassemblies and macroassemblies of giant unilamellar vesicles functionalized with a basic cellular machinery to express green fluorescent protein and specified neighbor-to-neighbor interactions. We show both that the local and programmable DNA pairing rules on the nanoscale are able to direct the microscale vesicles into macroscale soft matter assemblies and that the highly sensitive gene-expression machinery remains intact and active during multiple experimental steps. An in silico model recapitulates the experiments performed in vitro and covers additional experimental setups highlighting the parameters that control the DNA-directed bottom-up synthesis of higher-order selfassembled structures. The controlled assembly of a functional vesicle matrix may be useful not only as simplified natural tissue mimics but also as artificial scaffolds that could interact and support living cells.

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INTRODUCTION The specific and reversible self-assembly of modular building blocks hold great promise for becoming the foundation of tomorrow’s technology.1 Artificial vesicles feature an aqueous compartment separated from aqueous surroundings by a closed membrane analogous in composition and dimension to natural cells. The spontaneous or induced association of lipid molecules into membrane sheets that close and form artificial vesicles2 is a result of nonspecific intermolecular forces and constitutes a primary self-assembly process.3 Such vesicles can undergo a secondary self-assembly into higher-order structures.3 The complexity of the higher-order structures in terms of number of populations of vesicles incorporated and architecture strongly depends on the character of the linking agent. Aggregation induced by the osmotic expansion of vesicles causes increased hydrophobic interactions that neither depend on the nature of the solute added nor are strongly affected by the membrane composition of the vesicles.3 Consequently, specificity is not possible. The addition of soluble external agents (e.g., polymers,4 calcium,5,6 and DNA oligonucleotides7) increases the specificity by limiting the aggregation process to vesicles of specific membrane composition. Ligand−receptor pairs such as biotin−streptavidin3 and electrostatic interactions8 limit the aggregates to binary assemblies of vesicles. The highest degree of complexity is reached when using linear single-stranded DNA (ssDNA) oligonucleotides anchored to the surface of the building blocks. © 2013 American Chemical Society

The programmable self-assembly of superstructures composed of more than two distinct entities has consequently attracted significant attention in nanotechnological applications.9,10 DNA oligonucleotides were shown to act as specific adhesive elements for the programmable in-solution assembly of sophisticated higher-order superstructures of hard11−17 and soft colloids such as vesicles,18−23 water-in-oil (w/o) emulsion droplets,24,25 and even natural cells.26 The ability of vesicles to confine, transport, and manipulate (bio)chemical cargo makes them not only the prevalent model for natural cells27 but also ideal as bioreactors,28 drug delivery systems,29 and the starting point for the synthesis of artificial cells.30 To develop minimal artificial cells, single vesicles were functionalized with artificial genetic programs and cellular components to synthesize proteins.31−34 Assemblies of vesicles have been realized to engineer bioreactors35 and were proposed as multicomponent or multifunctional drug delivery systems36 and as starting point for the synthesis of primitive cell communities.37 Here, we focus on the spatially controlled DNA-directed bottom-up synthesis of microassemblies and macroassemblies of gene-expressing giant unilamellar vesicles (GUVs) by employing both in vitro and in silico experiments. We found Received: July 11, 2013 Revised: November 13, 2013 Published: December 2, 2013 15309

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Table 1. Functionalization of the GUV Populations fluorescence GUV population 1 2 3 4 5 6 7 8

membrane functionalization

DNA sequence

lumen

Strept-AF488a: btn-ssDNA1b Strept-AF532: btn-ssDNA2 Strept-AF532: btn-ssDNA7 Strept-AF350: btn-ssDNA1 Strept-AF350: btn-ssDNA4 Strept-AF350: btn-ssDNA2 Strept-AF350: btn-ssDNA5 Strept-AF532: btn-ssDNA3 Strept-AF532: btn-ssDNA6 Strept-AF350: btn-ssDNA7 Strept-AF350: btn-ssDNA7

btn-TCGTGTGTAATCTTT-3′ btn-AAAGATTACACACGA-3′ btn-TGTACGTCACAACTA-3′ btn-TCGTGTGTAATCTTT-3′ btn-CATCCATGGTGGAGG-3′ btn-AAAGATTACACACGA-3′ btn-TGGAGGGCTCTTTCT-3′ btn-CCTCCACCATGGATG-3′ btn-AGAAAGAGCCCTCCA-3′ btn-TGTACGTCACAACTA-3′ btn-TGTACGTCACAACTA-3′

membrane

identifier in the in silico model, color attributed in Figure 7

greenc redd red bluee

W, green X, red

redf

blue

X, red

greeng

red

W, green Z, uncoloredh

red

blue blue

Y, gray

Streptavidin Alexa Fluor conjugate. bBiotinylated single-stranded DNA oligonucleotide. cGreen fluorescence from the streptavidin Alexa Fluor 488 conjugate. dRed fluorescence from the streptavidin Alexa Fluor 532 conjugate. eBlue fluorescence from the streptavidin Alexa Fluor 350 conjugate. f Red fluorescence from the encapsulation of Atto 565. gGreen fluorescence from the synthesis of the enhanced green fluorescent protein. hIn Figure 7, only assembled GUVs are colored. Because GUV population Z is excluded from the assembled structures, no color is attributed to this GUV population. a

osmotic pressure and that the sodium iodide concentrations were not affected by the adjustments. The change in osmolarity after the 120 min incubation period of the self-assembly experiments was quantified for six samples for each of the observation chamber designs (see below). For the functionalization of the GUV membrane surface, streptavidin Alexa Fluor 350, 488, and 532 conjugates from Invitrogen (Basel, Switzerland) were dissolved to a final concentration of 1 mg/ mL. 15-mer biotinylated ssDNA (btn-ssDNA) oligonucleotides were synthesized, modified, purified by HPLC, and dissolved to a final concentration of 100 μM by the supplier (Sigma-Genosys, Buchs, Switzerland); see Table 1 for the sequences of the btn-ssDNA oligonucleotides. Pairs ssDNA1-ssDNA2, ssDNA3-ssDNA4, and ssDNA5-ssDNA6 had a complementary sequence. GUV Preparation. The GUV preparation in microplates (U96 MicroWell plates, polystyrene clear, U-bottom, Thermo Fisher Scientific, Langenselbold, Germany) based on the w/o emulsion transfer method39,40 as well as the functionalization of GUVs with btnssDNA oligonucleotides is detailed elsewhere.21 Briefly, the microplate wells were filled with 100 μL of HS1, layered with 50 μL of PS, and incubated for 10 min at RT. To prepare the w/o emulsions, 20 μL of IS was added to 1 mL of PS. The mixtures were mechanically agitated, and 100 μL was added to each well. Centrifugation (3 min, 1500 g, RT) forced the emulsion droplets to cross the interface between HS1 and PS. Because of the density difference between HS1 and IS and the geometry of the well bottom, the resulting GUVs sedimented and formed a pellet in the center of the well. The PS was removed afterward by aspiration using a vacuum pump. Subsequent addition of 200 μL of HS1, centrifugation (3 min, 1500g, RT), and aspiration by a vacuum pump removed the remaining traces of the PS. GUV Membrane Functionalization. Streptavidin, btn-ssDNA oligonucleotides, and HS2 were incubated for 30 min at RT in a 1.35:0.5:98.15 volume ratio as indicated in Table 1. The media individually hosting the GUV populations were subsequently incubated with an equal volume of the preincubated mixtures of streptavidin and btn-ssDNA oligonucleotides. After a 1 h incubation, the GUVs were washed five times to remove excess linkers by pelleting, addition of new HS3, and resuspension. Self-Assembly. For the self-assembly process induced by centrifugation, equal volumes of the media hosting GUV populations 1 and 2 (or 1 and 3 as control) were transferred to a new well of a U96 microwell plate and gently mixed by aspiration. Centrifugation (3 min, 1500g, RT) forced the GUV to pellet in the center of the well. The pellets were transferred to a sealed observation chamber by gentle aspiration without resuspension.

the assembly process to be specific and the architecture of the assemblies to be DNA-guided, to be in good accordance with the in silico model, and to depend on the character of the aggregating forces (i.e., centrifugation, solute concentration gradient).

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EXPERIMENTAL SECTION

Materials. Phospholipids POPC (2-oleoyl-1-palmitoyl-sn-glycero3-phosphocholine) and DSPE-PEG2000-btn (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(poly(ethylene glycol))2000]) purchased in chloroform from Avanti Polar Lipids (Alabaster, AL, USA) were mixed in a molar ratio of 98:2. After chloroform evaporation (under vacuum, 60 min), light mineral oil (Sigma-Aldrich, Buchs, Switzerland) was added, resulting in a 200 μM final concentration of phospholipids. The phospholipid solution (PS) was then sonicated (30 min, 50 °C) using a Sonorex Digitec DT 156 BH (Bandelin GmbH, Berlin, Germany), followed by overnight incubation at room temperature (RT). The PS was protected from light, stored under a normal atmosphere at RT, and used within 1 week. The buffered external media hosting the GUVs, called hosting solution (HS), contained 12.5 mM Tris buffer at pH 8 with various glucose (HS1, 500 mM; HS2, 450 mM; HS3, 475 mM) and sodium iodide concentrations (HS1, 0 mM; HS2, 25 mM; HS3, 12.5 mM). Sodium iodide was reported by the authors to be most compatible with the GUV stability of all tested monovalent salts.38 Intravesicular solution (IS) 1 for GUV populations 1 to 4 and 7 contained 12.5 mM Tris buffer at pH 8 with 250 mM sucrose and 250 mM glucose. IS2 for GUV populations 5 and 8 additionally contained 1 μg/mL Atto 565 biotin (Sigma-Aldrich, Buchs, Switzerland) for red fluorescence. IS3 for gene-expressing vesicle population 6 contained the E. coli-based S30 T7 cell-free gene expression kit for circular DNA (Promega Corporation, Madison, WI, USA) with all amino acids (2 mM each), an S30 Premix (with rNTPs, tRNAs, an ATP-regenerating system, and salts), an S30 cellular extract including the bacteriophage T7 RNA polymerase, and a custom plasmid DNA pMW-T7eGFP (39 nM) with a T7 promoter. Sucrose (2 M) was added in a volume ratio of 12:1 (cell-free system/sucrose) prior to the encapsulation in population 6 GUVs. High-quality water (Milli-Q, Millipore, Brussels, Belgium) was used throughout the experiments. All aqueous media except for IS3 were filtered using a sterile vacuum-driven Millipore Express Plus membrane with 0.22 μm pores (Millipore, Brussels, Belgium) and stored at −20 °C until use. All ISs and HSs were osmotically adjusted using a vapor-pressure osmometer (Vapro5520, ELITech Group, Puteaux, France), making sure that the ISs and HSs were at the same 15310

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Figure 1. Binary macroassemblies induced by centrifugation. (a, b) Schematic representation of the connectivity of the GUV populations employed. (First and third columns) Fluorescence micrographs and (second and fourth columns) colocalization of the fluorescence signal for GUV populations 1 and 2 (left half) and populations 1 and 3 (right half). The membranes of GUVs of population 1 were fluorescently labeled green (streptavidin Alexa Fluor 488). The membranes of GUVs of populations 2 and 3 were fluorescently labeled red (streptavidin Alexa Fluor 532). Micrographs of the first and third columns represent blowups of the same section (dashed line). The scale bar applies to all micrographs and represents (c−f) 1000, (g− k) 500, (l−o) 200, and 20 μm (p−s). complete sealing of the chamber. After the transfer of 20 μL of the sample, the microscope slide was attached. For the sealed chamber design, two intact frame seals were fixed on top of each other, resulting in spacers of the same height as for the nonsealed chamber design. The samples were evaluated using a Nikon Eclipse TE2000-S inverted light and fluorescence microscope with a Nikon Intensilight light source and using a 2×, 4×, or 10× air objective or a 100× oil-immersion objective (Nikon). Micrographs were taken with a Photometrics Cascade II 512 camera and Micromanager open source microscopy software, version 1.4. The fluorescence micrographs were automatically contrast adjusted (equally across the entire image) using Adobe Photoshop CS5, version 12.0.4. Multiplication of the red and green channel by using the channel calculations function in Adobe Photoshop led to a measure of the colocalization of streptavidin Alexa Fluor 488 conjugate and streptavidin Alexa Fluor 532 conjugate in Figures 1−3. ImageJ (version 1.45s) was used for the cross-section

For the self-assembly process driven by a solute concentration gradient (i.e., osmotically driven), 30 μL of either a 1:1-mixture (v/v) of GUV population 1 and 2 (or 1 and 3 as control) or a 1:1:4 mixture (v/v) of GUV populations 4−6, populations 5−7, or populations 4, 6, and 8 were transferred to an Eppendorf tube, gently mixed by aspiration prior to the transfer (20 μL) to the nonsealed observation chamber (or to a sealed chamber as control), and incubated for 2 h at RT. The results were evaluated by fluorescence microscopy. Microscopy and Image Processing. To prevent GUVs from adhering to surfaces, microscope slides and 24 × 60 mm2 coverslips (Menzel Glaeser, Braunschweig, Germany) were treated with the hydrophobic PlusOne Repel-Silane ES (GE Healthcare, Hillerød, Denmark) according to the supplier’s recommendations. For the nonsealed chamber design allowing evaporation, manipulated frame seals (25 μL, Capitol Scientific Inc., Austin, TX, USA) acted as spacers of 0.5 mm height at the short edges of a coverslip in order to avoid 15311

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study in Figure 6b. Theoretical predictions of the fluorescence intensities in Figure 6b were calculated on the basis of the assumption of two spherical objects with the radius of the two GUVs depicted in Figure 6b and an integration of the fluorescence signal over the entire height. All figures were prepared using Adobe Illustrator CS5, version 15.0.2. The contours of the assembled GUVs in Figures 4 and 5 were traced manually using the pen tool of Adobe Illustrator. Size Distribution. The data for the size distributions were obtained in duplicates by capturing 50 fluorescence micrographs (0.156 μm/pixel) of GUV populations 4−6 in individual observation chambers. To reproduce the GUV concentrations used for the selfassembly experiments, populations 4 and 5 were mixed in a 1:5 (v/v) ratio, and population 6 was mixed in a 2:1 (v/v) ratio with HS1 prior to the transfer to the observation chambers. The object diameters in the 300 micrographs were measured using a custom Matlab algorithm (Matlab R2012a 7.14.0.739, Mathworks, Natwick, MA, USA). Image preprocessing was performed by the algorithm in order to darken the complex background and enhance the contrast using top-hat filtering with a large discoid structuring element, contrast adjustment, and a morphological closing of the image. Each image was then subject to a circular Hough transform, which detected circular shapes larger than 10 pixels (i.e., 1.56 μm) in radius. This enabled the automated detection of GUVs with high degrees of overlap. Finally, each GUV was inspected manually to remove any erroneously detected circles, typically at sites where many small GUVs were clustered. Any distinguishable GUVs missed by the algorithm were likewise added manually. Objects smaller than 10 pixels in radius were only rarely sharp/bright enough to enable measurement. For the histograms in Figure 6a, the Freedman−Diaconis rule was applied to determine the optimal bin sizes for each GUV population independently.41 In Silico Model. A 2D square toroidal lattice (40 × 40) with degree k = 8 was filled with randomly positioned GUVs of two populations (i.e., populations W and X (scenario A)) or three populations (i.e., populations W, X, and Y (scenario B) or populations W, X, and Z (scenario C)). For each scenario, five replicas were prepared. Population W represents in vitro populations 1 and 6, population X represents in vitro populations 2 and 5, population Y represents in vitro population 4, and population Z represents in vitro population 7. The concentrations of population W varied between 0.625 and 50% in eight distinct steps as indicated in the category labels of Figure 8. The remaining nodes were filled with GUVs of population X (scenario A), populations X and Y in equal numbers (scenario B), or populations X and Z in equal numbers (scenario C). In summary, 120 samples (i.e., three scenarios times eight different ratios times five replicas) were evaluated. In scenarios A and B, the GUV populations were mutually complementary; in scenario C, population Z was excluded from the assemblies because they were defined as noncomplementary. A custom Matlab algorithm was then used to define assembled GUVs (i.e., nodes were defined as assembled if one of the eight neighboring nodes was occupied by a complementary GUV). This procedure was recursively repeated until all GUVs were found that made up one assembly. For each scenario and concentration, the mean of the five replicas for (i) the mean number of clusters, (ii) the mean size of the clusters normed by the available number of nodes (i.e., 1600), and (iii) the mean number of GUVs linked to a GUV of population W were calculated. The mean standard deviations were calculated by extracting the root of the averaged sum of the squared standard deviations. The minimal cluster size was two. Clusters smaller than 1% of the largest cluster in the same replica were discarded from the analyses.

poly(ethylene glycol) (PEG) tethers42 and fluorescently labeled streptavidin as anchors. The PEG tethers are known not only to prevent the nonspecific aggregation of lipid surfaces driven by attractive van der Waals and other less pronounced aggregating potentials such as polymer bridging and the hydrophobic effect42 but also to provide high detachment resistance43 and to show no detectable intermembrane transfer of linkers from donor vesicles to acceptor vesicles.44 Exploiting the noncovalent biotin−streptavidin binding instead of incorporating DNA oligonucleotides covalently attached to a large hydrophobic group45 promotes the use of modular parts and will ease the reuse of the components46 in future studies. Estimate of the Linkage Parameters. Linkage-induced receptor accumulation between vesicles47−50 is of particular interest in vesicle self-assembly because of its potential to selfterminate the assembly process by linker depletion and therefore to determine the coordination number of vesicles3 as a function of the surface linker density.51,52 However, here we did not intend the linker depletion to limit the dimensions of the assembled GUV structures. We consequently doubled the concentration of the biotinylated PEG tethers compared to that in our previous study.21 Following our previous discussion24 and considering the arguments of Larsson et al.,53 we estimate the DNA surface coverage of the GUVs to be 15 200 to 30 400 strands/μm2 and the average projected area available to a tethered biotin−streptavidin complex to be 6580 Å2. This number is in accordance with the experimental results and theoretical considerations of Burridge et al.,43 who showed almost complete surface coverage for 2 mol % DSPE-PEG2000btn and calculated the area that the PEG tethers and streptavidin will sample to be approximately 8330 Å2. Thus, we assume almost complete streptavidin surface coverage of the GUVs used in this study. Binary Macroassemblies Induced by Centrifugation. For the first step, we demonstrated the basic principle of the assembly system: formation of macroscale GUV assemblies from two different GUV populations without gene expression. When the GUVs were centrifuged during assembly, the populations form macroscopic pellets visible by eye. After the transfer to the observation chambers, the structural integrity of these macroscopic pellets was conserved only if the btn-ssDNA oligonucleotides of the two GUV populations (i.e., 1 and 2) were of complementary sequences, indicating proper assembly (Figure 1c). In the case of control noncomplementary GUV populations (i.e., 1 and 3), the vesicles were found to be solitary and randomly distributed over the surface of the observation chamber (Figure 1r). This finding supports both the specificity of the DNA-mediated self-assembly process and the absence of aggregating potentials not based on DNA hybridization such as streptavidin, polymer bridging, and the hydrophobic effect and is in agreement with our previous results.21,24 A multilayered structure was found for the assembled GUVs (Figure 1p). The multiplication of the fluorescence signal of the two differently labeled streptavidin populations (i.e., green and red) used to functionalize the membrane of the two GUV populations quantifies the colocalization of the two streptavidin populations as a result of adhesion. For the structures on the left half of Figure 1, the persistent signal of colocalized streptavidin populations (Figure 1d,h,m,q) indicates large GUV assemblies. On the right half of Figure 1, the decrease in the signal intensity for the colocalization of the fluorescence signal of the two streptavidin populations (Figure 1f,k,o,s) supports the absence of aggregated structures. However, the multilayered

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RESULTS AND DISCUSSION The w/o emulsion-transfer method employed in this study ensured the unilamellarity of the vesicles prepared39,40 and enabled the encapsulation of an E. coli-based cell-free geneexpression system31 with an expression plasmid to synthesize enhanced green fluorescent protein (eGFP). The membranes of the GUVs were functionalized with btn-ssDNA oligonucleotides employing phospholipids grafted with flexible biotinylated 15312

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Figure 2. Osmotically driven binary microassemblies. (a, b) Schematic representation of the functionalization and the connectivity of the GUV populations employed. (First and third columns) Fluorescence micrographs and (second and fourth columns) colocalization of the fluorescence signal for GUV populations 1 and 2 (left half) and populations 1 and 3 (right half). The membranes of GUVs of population 1 were fluorescently labeled green (streptavidin Alexa Fluor 488). The membranes of GUVs of populations 2 and 3 were fluorescently labeled red (streptavidin Alexa Fluor 532). Micrographs of the same section were taken before (c, e) and after (g, i) a 2 h incubation in the nonsealed observation chamber. Scale bar: 20 μm.

and the colocalization of the fluorescence signal of the streptavidin populations (cf. Figure 2h,k). We found an optimal incubation time of at least 2 h. Figure 3 shows a representative example of assembled vesicles after a prolonged incubation. We noticed the tendency of the assembled structures to assume an overall spherical shape. Although the contact areas of the assembled vesicles

architecture of the assembled GUVs prevents the data on the colocalization of the two streptavidin signals from being conclusive because the signal may not result only from the persistence of contact areas of aggregated GUVs but also may be confounded by the multilayered architecture of the assembled vesicles, where the two signals become spatially superimposed without aggregation. Binary Microassemblies Resulting from the Osmotically Driven Assembly Process. To simplify the characterization of the vesicle assembly, we focused on 2D assembly architectures to demonstrate the principles of our system. To ensure a singlelayered architecture of the assembled GUVs, we changed the methodology to bring GUVs into close contact. Nardi et al.54 reported the transformation of entropic osmotic energy into mechanical motion for GUVs in persistent solute concentration gradients. To recreate such a concentration gradient in a simple observation chamber, we intentionally chose a nonsealed design55 to host the self-assembly experiments. The evaporation of water along the border of the sample resulted in a centric concentration gradient and induced both a net central flow of solutes and an osmotic pumping out of the GUVs causing the vesicles to move through the recoil produced.54 Consequently, GUVs accumulated in the center of the sample droplet during the 2 h incubation period (cf. Figure 2c,g). Furthermore, the increase in osmotic pressure of the hosting medium caused the GUVs to shrink through water permeation across the bilayer. The resulting surplus of surface area was compensated for by deviations from the spherical shape. Depending on the complementarity of the btn-ssDNA oligonucleotides vesicles were found either to be ellipsoid (Figure 2i) or to form extensive contact areas with adjacent GUVs (Figure 2g). These so-called adhesion plaques56 were easily detectable by the straight contact area (cf. Figure 2g,i)

Figure 3. Formation of extensive contact areas. (a) Schematic representation of the functionalization and the connectivity of the GUV populations employed. (b) Fluorescence micrograph and (c) colocalization of the fluorescence signal for GUV populations 1 and 2 after a 6 h incubation in the nonsealed observation chamber. The membranes of GUVs of population 1 were fluorescently labeled green (streptavidin Alexa Fluor 488). The membranes of GUVs of population 2 were fluorescently labeled red (streptavidin Alexa Fluor 532). Scale bar: 20 μm. 15313

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Figure 4. Ternary microassemblies incorporating gene-expressing GUVs. (a, f) Schematic representation of the functionalization and the connectivity of the GUV populations employed. (b−d, g−i) Fluorescence micrographs. (b, g) The membranes of GUVs of populations 4 and 5 were fluorescently labeled blue (streptavidin Alexa Fluor 350). (c, h) The lumen (Atto 565) of population 5 and the membranes (streptavidin Alexa Fluor 532) of GUVs of population 6 were fluorescently labeled red. (d, i) Enhanced green fluorescent protein was synthesized in the lumen of GUV population 6. (e, k) Scheme of the ternary assemblies with colors indicating the signals of the fluorescent markers used for the three GUV populations. Only assembled GUVs were contoured manually. The mutual complementarity of the three pairs of btn-ssDNA oligonucleotides with which the membranes of the three GUV populations were functionalized ensured unrestricted growth of the aggregates during the self-assembly process, resulting in large ternary assemblies. Differences in the packing density and the deviation from the circular shape are caused by the differences in the osmotic pressure of the hosting media during the incubation in the nonsealed (first row) and sealed (second row) observation chamber. Scale bar: 20 μm.

Figure 5. Binary microassemblies incorporating gene-expressing GUVs. (a, f) Schematic representation of the functionalization and the connectivity of the GUV populations employed. (b−d, g−i) Fluorescence micrographs. The membranes of GUVs of (b) populations 5 and 7 and (g) populations 4 and 8 were fluorescently labeled blue (streptavidin Alexa Fluor 350). The lumen (Atto 565) of populations (c) 5 and (h) 8 and the membranes (streptavidin Alexa Fluor 532) of GUVs of (c, h) population 6 were labeled red. (d, i) Enhanced green fluorescent protein was synthesized in the lumen of GUV population 6. (e, k) Scheme of the binary assemblies with colors indicating the signals of the fluorescent markers used for the three GUV populations. Only assembled GUVs were contoured manually. Because of the exclusion of either GUV population 7 (first row) or population 8 (second row), GUVs of population 6 were surrounded exclusively by GUV population (e) 5 or (k) 4. The self-assembly process was self-terminating, resulting in binary assemblies of limited extension. (k) The arrow indicates a GUV of population 4 interlinking two assemblies. Scale bar: 20 μm.

streptavidin Alexa Fluor conjugate (i.e., Alexa Fluor 350) to anchor the btn-ssDNA oligonucleotides to the membrane of GUV populations 4, 5, 7 and 8 (cf. Table 1). This enabled us to keep track of the self-assembly process (blue and red channel) and the gene expression activity (green channel) simultaneously. Assemblies were detected visually through the elastic deformations of the assembled vesicles (i.e., deviation from the spherical shape) that proved to be reliable in the previous experiments (cf. Figure 2g) and that are commonly used to

became extensive, the distance between the assembled structures increased during the incubation process. Binary and Ternary Microassemblies Composed of GeneExpressing GUVs Resulting from the Osmotically Driven Assembly Process. In the final step, we focused both on the control of the architecture of the assembled structures and the increase in the complexity of the assemblies by introducing a third GUV population that was functionalized to participate in the DNA-guided self-assembly process and that in addition was able to express a reporter gene. We decided to employ the same 15314

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distinguish assembled and nonassembled soft colloidal particles.3,5,6 Figure 4b−e shows a representative example of large ternary assemblies composed of gene-expressing GUVs resulting from the mutual complementarity of the three pairs of btn-ssDNA oligonucleotides on the three GUV populations. The limited extension of the structures in Figure 2g, which were also predicted to grow unrestrictedly (cf. Figure 7b−e and discussion below) indicates that the ternary structures are of only limited extension. Consequently, we term them ternary microassemblies. The E. coli-based cell-free system entrapped in GUV population 6 proved to be stable and functional during several GUV manipulation steps and under increased osmotic pressure. To analyze the influence of osmotic shrinkage on the assembly process and the gene expression activity, we minimized the change in osmotic pressure (3.5 ± 1.0% for the sealed vs 23.7 ± 1.7% for the nonsealed design, with standard deviations as errors) by using a sealed observation chamber. Figure 4g−k reveals a looser packing of the GUVs in the assemblies and less pronounced adherent contacts. However, the extension of the aggregates was comparable to the assemblies in Figure 4b−e. A qualitative comparison between the strength of the fluorescence signal in Figure 4d,i reveals no considerable difference in the gene-expression activity as a function of the osmotic shrinkage of the GUVs. The spatial arrangement and interconnectivity of cells represents a critical element of their higher-order function in tissues. However, the in vitro emulation of these architectural aspects is often difficult or impossible.26 With the goal of terminating the self-assembly process and thereby limiting the lateral dimension of the gene-expressing GUV assemblies and defining the GUV−GUV contacts, we analyzed the selfassembly process if one of the non-gene-expressing GUV populations was functionalized with btn-ssDNA oligonucleotides noncomplementary to both other GUV populations. Unlike the large binary structures of assembled GUVs in Figure 2g, we found the binary gene-expressing microassemblies of Figure 5 to be defined in the architecture. The self-assembly process self-terminated after the gene-expressing GUVs were surrounded by a single layer of nonexpressing GUVs. Only rarely was a connection between two microassemblies found (cf. arrow, Figure 5k). Characterization of the GUVs That Compose the Microassemblies. After the assembly experiments, GUV populations 4−6 were characterized for vesicle concentration and size distribution from 50 micrographs (n = 5053 for population 4, 5005 for population 5, and 550 for population 6). With a total area of 3.2 × 105 μm2 for the 50 micrographs taken and a height of the two frame seals of 500 μm, populations 4 and 5 had an estimated concentration of about 31 000 vesicles per microliter; with a concentration of about 3400 vesicles per microliter, population 6 was almost an order of magnitude less concentrated. In addition to the concentration, the characteristics of the size distribution of population 4 and 5 GUVs were almost identical (Figure 6a); however, the size distribution of the gene-expressing population 6 GUVs differed considerably from those of the other two populations. This difference can be attributed to GUV preparation and functionalization that required the intravesicular medium to have a higher density than the external medium in order to induce GUV sedimentation during centrifugation.21 The gravitational force inducing sedimentation depends on the volume of the body, whereas the drag force opposing sedimentation depends on the

Figure 6. Characterization of GUV populations 4−6. (a) Histogram of the size distribution of the three GUV populations. The mean diameters with standard deviations were 5.03 ± 1.96 μm for population 4 (n = 5053), 5.86 ± 2.36 μm for population 5 (n = 5005), and 9.24 ± 3.14 μm for population 6 (n = 550). (b) Crosssection intensities (solid line) of the fluorescent streptavidin Alexa Fluor 532 conjugate (left) and enhanced green fluorescent protein (right) signal in a representative micrograph of two GUVs of population 6 along the center line. Dashed lines represent theoretical predictions of the fluorescence intensities for two spheres with a radius of the two GUVs. Scale bar: 20 μm.

projected area. Consequently, the lower limit for the radius of GUVs sedimenting positively correlates with the density difference. “Ballasting” the E. coli-based cell-free system of population 6 GUVs by the addition of sucrose both increased the vesicle yield and decreased the mean diameter for this GUV population and consequently harmonized the yield and size distribution of all GUV populations (data not shown). However, we refrained from a complete harmonization because the additional sucrose in population 6 GUVs diluted the cellfree system, which entailed a reduced eGFP fluorescence signal. In addition to the size distribution, the fluorescence signal of population 6 GUVs was analyzed (Figure 6b). The crosssectional intensity revealed that the signal of the fluorescent streptavidin employed to functionalize the GUV surface with btn-ssDNA oligonucleotides was found exclusively on the membrane, whereas the fluorescence signal of the water-soluble eGFP expressed in the GUV lumen was found to follow the theoretical predictions both in the intensity values and in shape. Because of the small total volume of all of the GUVs expressing eGFP, analytical methods of quantifying the eGFP concentration were not available. Modeling the Size Distribution and Stoichiometry of the GUV Assemblies. The resulting distribution of assemblies with varying type and quantities of GUVs was investigated in a 2D in silico model. We were particularly interested in 15315

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Figure 7. Binary and ternary assemblies of modeled GUVs. (a, f, l) Schematic representation of the connectivity of the GUV populations employed. The square lattice was filled with randomly positioned GUVs of (b−e, scenario A) two mutually complementary populations (W, X), (g−k, scenario B) three mutually complementary populations (W, X, Y), (m−p, scenario C) or three GUV populations (W, X, Z), of which only two were complementary (W, X) and consequently could undergo aggregation. For each scenario, eight different ratios of the GUV populations were tested, four of which are depicted in the panels as indicated. (b−e, g−k, and m−p) Assembled GUVs are indicated as green (W), red (X), and gray (Y); uncolored cells represent GUVs either not assembled (b−e, g−k) or from population Z (m−p). Only GUVs of different colors became directly linked; neighboring GUVs of the same color are linked only indirectly. (e) Because of the toroidal architecture of the lattice, GUVs can become linked to neighbors at the opposite edge; the arrow indicates one example.

understanding the influence of the small number of geneexpressing GUVs that we found in the in vitro experiments and the presence of a third population of GUVs not participating in the self-assembly process. Both factors may have influenced the self-termination of the growth of the microassemblies and therefore their architecture. We modeled the binary and ternary self-assembly process for three different scenarios and eight different ratios of GUV populations. Briefly, the populations and the pairing rules were W, X, complementary (scenario A); W, X, Y, complementary (scenario B); and W, X, complementary, Z, noncomplementary (scenario C). The in silico model covered 24 experimental setups including the 3 setups performed in vitro: setup A with relative concentrations of 50% W and 50% X represents the binary macroassemblies of two equally concentrated GUV populations (cf. Figures 1, 2, and 7a); setup B (W, 5%; X, 47.5%; Y, 47.5) represents the ternary assemblies of three mutually complementary GUV populations (cf. Figures 4 and 7i); setup C (W, 5%; X; 47.5%; Z, 47.5) represents the binary assemblies of two complementary GUV populations in the presence of a third noncomplementary GUV population (cf. Figures 5 and 7o). The outcome of scenario B was as expected: independent of the relative frequencies of three mutually complementary GUV populations, there was one large cluster (Figure 8a) including all or almost all available GUVs (Figure 8b). The mean number of GUVs linked to one GUV of population W (Figure 8c) started with four for population W occupying 50% of all available nodes and approached eight when the neighboring of

one GUV of population W with another GUV of population W becomes less likely (i.e., with decreasing concentration of population W GUVs). With respect to the in vitro experiments, this indicates that the growth of the assemblies of Figure 4 is unrestricted as long as GUVs are available and no demixing of the GUV populations occurs (e.g., by slower or faster central movement of one of the GUV populations). A comparison of scenarios A and C, however, is more interesting. In both cases, two GUV populations assembled; the scenarios differed in the (A) absence and (C) presence of one GUV population not participating in the assembled structures. For high percentages of population W (i.e., 50, 33%), there is a small number of clusters (Figure 8a); for low percentages of population W (i.e., 2.5, 1.25, 0.625%), the number of clusters (Figure 8a) is defined by the number of available population W GUVs. For both regimes, the size of the clusters and the number of GUVs linked to one GUV of population W is half for scenario C compared to that for scenario A. The interpretation of this result is obvious as population W GUVs in scenario A are solely surrounded by complementary GUVs of population X, whereas GUVs of population W are neighboring equal numbers of complementary population X and noncomplementary population Z GUVs. Combined, we found no or almost no effect of the presence of a third GUV population not participating in the self-assembly process for regimes characterized by either high or low percentages of one GUV population. However, the outcome is different for intermediate percentages (i.e., 20, 10, and 5%). The mean number of clusters (Figure 8a) and as a consequence thereof 15316

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centrifugation (cf. Figures 1c and 7b) and on the binary microassemblies composed of gene-expressing GUVs. In particular, we found the ratio of 5% for population 6 GUVs versus 47.5% for populations 4 and 5 to be optimal for the selftermination of binary microassemblies during the in vitro studies (cf. Figure 5) and to be within the range predicted by the model to result in solitary microassemblies (cf. Figure 7o). Future studies may additionally focus on the surface DNA density and the oligonucleotide sequence complexity that were found along with the density of the entities to allow direct control of the size distribution and stoichiometry of assembled products for the DNA-mediated self-assembly process of natural cells.26

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CONCLUSIONS We demonstrated the DNA-directed bottom-up synthesis of biomimetic binary and ternary microassemblies and macroassemblies composed of GUVs with diverse functionalizations. Our results reveal that both the GUV membrane functionalization and the highly sensitive gene-expression machinery remained intact and active during multiple experimental steps. This indicates assemblies of functionalized GUVs to be suitable as new building blocks for novel functional biomaterials and tissue-engineering approaches. Future studies may further increase the complexity of the presented system by interfacing the DNA-directed microassemblies and macroassemblies of GUVs with the DNA-directed microassemblies of living cells.26 In this context, DNA oligonucleotides may provide a common “programming language” for the synthesis of a novel generation of scaffolds for tissue-engineering approaches57,58 in which living cells are implanted or “seeded” into an artificial scaffold composed of gene-expressing GUVs. Future studies will also have to focus on the engineering of communication networks to implement cooperative tissue mimics composed of cellmimicking artificial vesicles that have so far been implemented only for more robust soft colloid structures.59

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +41 44 632 44 78. E-mail: [email protected]. ch. *Phone: +81 0 761 51 5800. E-mail: [email protected]

Figure 8. Summary statistics for the binary and ternary assemblies of modeled GUVs: (a−c, black, scenario A) binary assemblies of GUVs of two mutually complementary populations (W, X); (dark gray, scenario B) ternary assemblies of three mutually complementary populations (W, X, Y); light gray (scenario C) three GUV populations (W, X, Z), of which only two are complementary (W, X) and consequently could undergo aggregation.

Present Address ∥

Swiss Federal Institute of Technology, Department of Chemistry and Applied Biosciences, Zurich, Switzerland. Author Contributions

M.H. and E.B. contributed equally to this work. The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript.

the mean cluster size (Figure 8b) differs remarkably for the two scenarios: clusters are 2 to 11 times more abundant and 3 to 22 times smaller if the assemblies are shielded from growth by the presence of a population not participating in the assemblies. Our in silico results show that the size distribution and stoichiometry of microassemblies can be controlled by the addition of a third population not participating in the selfassembly process (i.e., population Z). Although the in silico model is simple and considers neither the differences in sizes of the GUVs that make them susceptible to different numbers of adjacent vesicles nor the dynamic rearrangements that take place while the GUVs accumulate, it is in qualitative accordance with the experimental results on the self-assembly of the large macroscopic assemblies induced by

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The experimental work was performed at the Center for Fundamental Living Technology (FLinT) at the University of Southern Denmark. We thank J. Fredens and M. G. Jørgensen from the Department of Biochemistry and Molecular Biology, University of Southern Denmark, for technical assistance. The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007−2013) under grant agreement no. 249032 (project 15317

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acronym MATCHIT). M.H. was supported by the Swiss National Science Foundation (SNSF). E.B. was supported by the Dynamical Micro-Scale Reaction Environment (ERATO) project, Japan. D.D.L. has been partially supported by the EU project THEGRAIL under grant agreement no. 278557.

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