Water‐in‐Water Droplets Selectively Uptake Self‐Assembled DNA Nano/Microstructures: a Versatile Method for Purification in DNA Nanotechnology
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Abstract DNA is an excellent material for constructing self‐assembled nano/microstructures. Owing to the widespread use of DNA as a building block in laboratories and industry, it is desirable to increase the efficiency of all steps involved in producing self‐assembled DNA structures. One of the bottlenecks is the purification required to separate the excess components from the target structures. This paper describes a purification method based on the fractionation by water‐in‐water (W/W) droplets composed of phase‐separated dextran‐rich droplets in a polyethylene glycol (PEG)‐rich continuous phase. The dextran‐rich droplets facilitate the selective uptake of self‐assembled DNA nano/microstructures and allow the separation of the target structure. This study investigates the ability to purify DNA origami, DNA hydrogels, and DNA microtubes. The W/W‐droplet fractionation allows the purification of structures of a broad size spectrum without changes to the protocol. By quantifying the activity of deoxyribozyme‐modified DNA origami after W/W‐droplet purification, this study demonstrates that this method sufficiently preserves the accessibility to the surface of a functional DNA nanostructure. It is considered that the W/W‐droplet fractionation could become one of the standard methods for the purification of self‐assembled DNA nano/microstructures for biomedical and nanotechnology applications owing to its low cost and simplicity.Keywords:
DNA nanotechnology
The rational design of anisotropic interaction patterns is a key step for programming colloid and nanoparticle self-assembly and emergent functions. Herein, we demonstrate a concept for harnessing the capabilities of 3D DNA origami for extensive supracolloidal self-assembly and showcase its use for making truly monodisperse, patchy, divalent nanocuboids that can self-assemble into supracolloidal fibrils via programmable DNA hybridization. A change in the number of connector duplexes at the patches reveals that multivalency and cooperativity play crucial roles to enhance superstructure formation. We further show thermal and chemical switching of the superstructures as the first steps toward reconfigurable self-assemblies. This concept lays the groundwork for merging monodisperse 3D DNA origami, featuring programmable patchiness and interactions, with nanoparticle self-assembly.
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DNA nanotechnology
Cooperativity
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The relatively weak mechanical properties of hydrogels remain a major drawback for their application as load-bearing tissue scaffolds. Previously, we developed cell-laden double-network (DN) hydrogels that were composed of photocrosslinkable gellan gum (GG) and gelatin. Further research into the materials as tissue scaffolds determined that the strength of the DN hydrogels decreased when they were prepared at cell-compatible conditions, and the encapsulated cells in the DN hydrogels did not function as well as they did in gelatin hydrogels. In this work, we developed microgel-reinforced (MR) hydrogels from the same two polymers, which have better mechanical strength and biological properties in comparison to the DN hydrogels. The MR hydrogels were prepared by incorporating stiff GG microgels into soft and ductile gelatin hydrogels. The MR hydrogels prepared at cell-compatible conditions exhibited higher strength than the DN hydrogels and the gelatin hydrogels, the highest strength being 2.8 times that of the gelatin hydrogels. MC3T3-E1 preosteoblasts encapsulated in MR hydrogels exhibited as high metabolic activity as in gelatin hydrogels, which is significantly higher than that in the DN hydrogels. The measurement of alkaline phosphatase (ALP) activity and the amount of mineralization showed that osteogenic behavior of MC3T3-E1 cells was as much facilitated in the MR hydrogels as in the gelatin hydrogels, while it was not as much facilitated in the DN hydrogels. These results suggest that the MR hydrogels could be a better alternative to the DN hydrogels and have great potential as load-bearing tissue scaffolds.
Gelatin
Gellan gum
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While the self-assembly of different types of DNA origami into well-defined complexes could produce nanostructures on which thousands of locations can be independently functionalized with nanometer-scale precision, current assembly processes have low yields. Biomolecular complex formation requires relatively strong interactions and reversible assembly pathways that prevent kinetic trapping. To characterize how these issues control origami complex yields, the equilibrium constants for each possible reaction for the assembly of a heterotetrameric ring, the unit cell of a rectangular lattice, were measured using fluorescence colocalization microscopy. We found that origami interface structure controlled reaction free energies. Cooperativity, measured for the first time for a DNA nanostructure assembly reaction, was weak. Simulations of assembly kinetics suggest assembly occurs via parallel pathways with the primary mechanism of assembly being hierarchical: two dimers form that then bind to one another to complete the ring.
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This minireview discusses the advantages and challenges in constructing bioinspired double-network hydrogels mimicking the structure and/or properties of biological tissue.
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Research on hydrogels has been geared toward biomedical applications from the beginning due to their relatively high biocompatibility. Initially only the hydrophilic nature and the large swelling properties of hydrogels was explored. Continued research on hydrogels has resulted in the development of new types of hydrogels, such as environment-sensitive hydrogels, thermoplastic hydrogels, hydrogel foams, and sol-gel phase-reversible hydrogels. Application of hydrogels ranges from biomedical devices to solute separation. Examples of hydrogel applications in pharmaceutics, biomaterials, and biotechnology are briefly described.
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The relatively weak mechanical properties of hydrogels remain a major drawback for their application as load-bearing tissue scaffolds. Previously, we developed cell-laden double-network (DN) hydrogels that were composed of photocrosslinkable gellan gum (GG) and gelatin. Further research into the materials as tissue scaffolds determined that the strength of the DN hydrogels decreased when they were prepared at cell-compatible conditions, and the encapsulated cells in the DN hydrogels did not function as well as they did in gelatin hydrogels. In this work, we developed microgel-reinforced (MR) hydrogels from the same two polymers, which have better mechanical strength and biological properties in comparison to the DN hydrogels. The MR hydrogels were prepared by incorporating stiff GG microgels into soft and ductile gelatin hydrogels. The MR hydrogels prepared at cell-compatible conditions exhibited higher strength than the DN hydrogels and the gelatin hydrogels, the highest strength being 2.8 times that of the gelatin hydrogels. MC3T3-E1 preosteoblasts encapsulated in MR hydrogels exhibited as high metabolic activity as in gelatin hydrogels, which is significantly higher than that in the DN hydrogels. The measurement of alkaline phosphatase (ALP) activity and the amount of mineralization showed that osteogenic behavior of MC3T3-E1 cells was as much facilitated in the MR hydrogels as in the gelatin hydrogels, while it was not as much facilitated in the DN hydrogels. These results suggest that the MR hydrogels could be a better alternative to the DN hydrogels and have great potential as load-bearing tissue scaffolds.
Gelatin
Gellan gum
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