DNA cytoskeleton for stabilizing artificial cells

Abstract

Cell-sized liposomes and droplets coated with lipid layers have been used as platforms for understanding live cells, constructing artificial cells, and implementing functional biomedical tools such as biosensing platforms and drug delivery systems. However, these systems are very fragile, which results from the absence of cytoskeletons in these systems. Here, we construct an artificial cytoskeleton using DNA nanostructures. The designed DNA oligomers form a Y-shaped nanostructure and connect to each other with their complementary sticky ends to form networks. To undercoat lipid membranes with this DNA network, we used cationic lipids that attract negatively charged DNA. By encapsulating the DNA into the droplets, we successfully created a DNA shell underneath the membrane. The DNA shells increased interfacial tension, elastic modulus, and shear modulus of the droplet surface, consequently stabilizing the lipid droplets. Such drastic changes in stability were detected only when the DNA shell was in the gel phase. Furthermore, we demonstrate that liposomes with the DNA gel shell are substantially tolerant against outer osmotic shock. These results clearly show the DNA gel shell is a stabilizer of the lipid membrane akin to the cytoskeleton in live cells.iposomes have been used as artificial cell models to understand cell shape, membrane protein function, and lipid− protein interaction, among other biological functions (1-3). In addition, liposomes have been used as a platform for biosensing and as drug delivery systems (DDS) because of their excellent biocompatibility and biodegradability (4). However, liposomes collapse easily against environmental shifts and mechanical forces because of their low bending modulus. The fragility of liposomes causes uncontrolled leakage of the entrapped compounds and thus inhibits their use in biomedical applications and artificial cells experiments.In contrast, cell membranes are tolerant against environmental shifts and mechanical forces. The stability of cell membrane arises from the cytoskeleton underneath the membrane. The major component of cytoskeletons is actin (5). Actin gels show high elasticity (6), which ensures the stability of cell membranes against various forces. For liposomes, the use of actin filaments as a cytoskeleton is not an optimal strategy for the following three reasons: First, although actin bundles and actomyosin rings have been reconstituted in artificial cells (7,8), formation of an actin cortex underneath artificial membranes has been still challenging. Second, actin is hard to modify by chemical and genetic means because of its essentiality for cell growth. Third, the physicochemical properties of actin gels are still unclear (9, 10). Hence, the cytoskeleton of liposomes should be constructed with defined and designable materials. To accomplish this aim, DNA nanotechnology, which uses limited components with high designability in a nanometer scale (11), is a feasible candidate to construct cytoskeleton structures in artificial cells.DNA nanostructure...

Hybrid Soft Nanomaterials Composed of DNA Microspheres and Supramolecular Nanostructures of Semi‐artificial Glycopeptides

“…The programmable sequences and predictable interactions of DNA allow a variety of DNA nanostructures with controlled sizes and shapes to be constructed . Moreover, amphiphilic lipid‐based vesicles have been interlaced with the DNA nanostructures to construct, for example, active nanopores spanning the lipid bilayer membranes of vesicles or to control the stability, size, and morphology of vesicles. This clearly demonstrates the orthogonal coexistence of DNA nanostructures and lipid‐based vesicles.…”

Biophysical characterization reveals the similarities of liposomes produced using microfluidics and electroformation

“…Other potential methods to yield GUVs of arbitrary compositions involve stabilizing the membrane mechanically using nanostructures. Since OLA allows for the efficient encapsulation of substances in the interior of the vesicles, as well as coating from the exterior in well-defined conditions (18), an artificial cytoskeleton could be applied to the membrane, such as a DNA cytoskeleton (57).…”

Bottom‐Up Assembly of DNA–Silica Nanocomposites into Micrometer‐Sized Hollow Spheres

“…For example, Spatz and co‐workers have recently reported on the assembly of biofunctionalized droplets of water‐in‐oil emulsions using gold nanoparticles as mechanical stabilizer of the oil/water interface . In the field of DNA nanotechnology, Kurokawa and co‐workers recently demonstrated that the stability of batch‐produced, cell‐sized liposomes and droplets can be enhanced by assembling Y‐shaped DNA oligomers on the inner surface of cationic lipid membrane vesicles to form a thin polymeric DNA shell that increases the interfacial tension, elastic modulus, and shear modulus of the droplet surface . Inspired by these approaches and based on earlier investigations of the excellent biomolecular modifiability and biostability of DNA‐modified silica nanoparticles (DNA‐SiNPs), we wanted to investigate whether the self‐assembly of novel composite materials of DNA‐SiNPs and DNA polymers at the inner interface of microfluidic droplets is possible.…”

“…[9] In the field of DNAn anotechnology,K urokawa and co-workers recently demonstrated that the stability of batch-produced, cell-sized liposomes and droplets can be enhanced by assembling Y-shaped DNAo ligomers on the inner surface of cationic lipid membrane vesicles to form at hin polymeric DNAs hell that increases the interfacial tension, elastic modulus,a nd shear modulus of the droplet surface. [10] Inspired by these approaches and based on earlier investigations of the excellent biomolecular modifiability and biostability of DNA-modified silica nanoparticles (DNA-SiNPs), [11] we wanted to investigate whether the self-assembly of novel composite materials of DNA-SiNPs and DNA polymers at the inner interface of microfluidic droplets is possible.I ndeed, we found that polymerization of SiNPs capped with primers for clamped hybridization chain reaction (C-HCR) [12] gives ready access to an ovel class of composite materials that display material properties that are distinctively different from those of conventional DNAh ydrogels. We demonstrate that the new composites self-assemble underneath the interfacial layer of positively charged waterin-oil (W/O) droplets,t hus enabling the high-throughput fabrication of micrometer-sized hollow spheres that could be used for biomedical research.We initially investigated whether DNA-SiNPs can be converted into polymer composites using the C-HCR.…”