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WO2014078636A1 - Auto-assemblage d'hydrogel d'acides nucléiques - Google Patents

Auto-assemblage d'hydrogel d'acides nucléiques Download PDF

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Publication number
WO2014078636A1
WO2014078636A1 PCT/US2013/070260 US2013070260W WO2014078636A1 WO 2014078636 A1 WO2014078636 A1 WO 2014078636A1 US 2013070260 W US2013070260 W US 2013070260W WO 2014078636 A1 WO2014078636 A1 WO 2014078636A1
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Prior art keywords
hydrogel
nucleic acid
dna
shaped hydrogel
subunit
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Inventor
Peng Yin
Alireza Khademhosseini
Hao Qi
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Brigham and Womens Hospital Inc
Harvard University
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Brigham and Womens Hospital Inc
Harvard University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient

Definitions

  • the invention relates, in some embodiments, to the field of nucleic acid
  • Self-assembly refers to a process by which a disordered system of pre-existing components forms an organized structure as a consequence of specific local interactions among the individual components without external direction. Self-assembly processes are particularly useful in the field of nucleic acid nanotechnology. These processes take advantage of the molecular recognition properties of nucleic acids to create, for technological purposes, artificial, often intricate, nucleic acid structures.
  • a single hydrogel subunit is covalently conjugated to at least one nucleic acid (e.g., at least one nucleic acid concatemer).
  • the nucleic acid on each subunit acts as "glue” to bring together (i.e. , self- assemble) multiple hydrogel subunits in a controlled fashion.
  • a plurality of hydrogel subunits can be directed to self-assemble into single or multiple architecturally distinct, typically predetermined, structures.
  • shaped hydrogel subunits surface-modified with at least one nucleic acid referred to herein as a shaped nucleic acid hydrogel subunit.
  • the nucleic acid is a concatemer.
  • compositions comprising at least two shaped hydrogel subunits, each surface-modified with at least one nucleic acid, wherein the at least two shaped hydrogel subunits are joined to each other through sequence- specific hybridization between the nucleic acids.
  • the nucleic acid is a concatemer.
  • pluralities of shaped hydrogel subunits each surface-modified with at least one nucleic acid concatemer, wherein the shaped hydrogel subunits are joined to each other through sequence- specific hybridization between the nucleic acids.
  • the pluralities of shaped hydrogel subunits are specifically arranged to form three-dimensional structures.
  • a three- dimensional hydrogel structure comprising combining at least two shaped hydrogel subunits, each surface-modified with at least one nucleic acid concatemer, in an aqueous assembly system or in an interfacial assembly system, thereby providing for nucleic acid hybridization and shaped hydrogel subunit self-assembly.
  • methods of producing a shaped hydrogel subunit surface-modified with a nucleic acid comprising conjugating a nucleic acid to a polyethylene glycol (PEG) monomer to form nucleic acid-PEG-acrylate, combining the nucleic acid-PEG-acrylate with PEG-diacrylate and a photoinitiator to form a mixture, and exposing the mixture to ultraviolet light under a photomask to produce the shaped hydrogel subunit modified with at least one nucleic acid.
  • methods further comprise producing at least one concatemer through rolling circle amplification of the at least one nucleic acid.
  • the nucleic acid is a concatemer.
  • methods comprising delivering cells or other biomolecules to a site of interest in vivo using a shaped nucleic acid hydrogel subunit of the invention, a composition of the invention, or a plurality of shaped nucleic acid hydrogel subunits of the invention.
  • a shaped hydrogel subunit is a cube, a tube or a sphere.
  • a shaped hydrogel subunit is comprised of polyethylene glycol, gelatin methacrylate or alginate.
  • a shaped hydrogel subunit has a diameter of about 1 ⁇ to about 1 mm.
  • a nucleic acid concatemer is produced through rolling circle amplification (RCA).
  • a shaped hydrogel subunit is surface-modified with at least two nucleic acids. In some embodiments, at least two nucleic acids are different from each other and are present on different surfaces of the shaped hydrogel subunit. In some embodiments, a shaped hydrogel subunit is surface-modified with at least three nucleic acids.
  • the nucleic acids in some embodiments, are nucleic acid concatemers.
  • a shaped hydrogel subunit contains cells.
  • cells are stem cells, progenitor cells or differentiated cells.
  • FIG. 1 shows a process of a non-limiting example of stencil-based hydrogel fabrication and surface modification with nuclei acid concatemers
  • FIG. 2A shows schematics of a non-limiting example of a fabrication process of hydrogel cubes uniformly modified with DNA concatemers
  • FIGs. 2B-2D show a phase contrast image (FIG. 2B), a fluorescent image (FIG. 2C), and a scanning electron microscopy (SEM) image (FIG. 2D) of hydrogels carrying short 56-nucleotide (nt) single- stranded DNA primers
  • FIGs. 2E-2G show a phase contrast image (FIG. 2E), a fluorescent image (FIG. 2F), and a scanning electron microscopy (SEM) image (FIG. 2G) of amplified single- stranded DNA concatemers (the gels in (FIG. 2C) and (FIG. 2F) were stained with SYBR ® Gold before imaging)
  • FIG. 2A shows schematics of a non-limiting example of a fabrication process of hydrogel cubes uniformly modified with DNA concatemers
  • FIGs. 3A-3K show non-limiting examples of self-assembly of hydrogel cubes with uniform DNA concatemer modification
  • FIGs. 4A-4E show non-limiting examples of self-assembly of hydrogel subunits (e.g. , cubes) with face- specific DNA concatemer modifications;
  • FIG. 5A shows a schematic of a non-limiting example of hydrogel cube self-assembly at a liquid-liquid interface
  • hydrogel cubes were floated at the interface formed between aqueous phosphate buffered saline (PBS) liquid (upper) and fluorinert FC-40 liquid (bottom) and agitated with rotary shaking
  • FIG. 5B shows dimers formed from red and blue hydrogel cubes (left, schematics; right, stereomicroscopy image)
  • FIGs. 5C-5E show examples of four hydrogel cubes self-assembled into a chain (FIG. 5C), a T-junction (FIG. 5D) or a square (FIG. 5E) based on their surface DNA concatemer modification pattern (left, schematics; right, color stereomicroscopy image);
  • FIGs. 6A-6E show non-limiting examples of patterned DNA concatemer modification on the surface of shaped hydrogel cubes and DNA concatemer-directed hydrogel cube dimer formation (e.g. , self-assembly of two hydrogel cubes);
  • FIGs. 7A-7F show phase contrast (left) and fluorescent (right) images for non-limiting examples of DNA concatemer-modified cell-free hydrogel cubes (top), cell-encapsulated hydrogel cubes without DNA concatemers (middle), and cell-encapsulated hydrogel cubes modified with DNA concatemers (bottom) (DNA concatemers and cells fluoresce; scale bar, 100 microns);
  • FIG. 8A shows a single- stranded 84-nt DNA primer before amplification (middle lane) and a post- amplification DNA concatemer (right lane) on 1% agarose gel, stained with SYBR ® Gold (left lane, 100 bp DNA ladder with the 2072 bp fragment labeled);
  • FIG. 8B shows DNA concatemers amplified from a 84-nt primer tethered on a PEG surface (the DNA concatemers were stained with SYBR ® gold);
  • FIG. 8C shows a scanning electron microscopy (SEM) image of amplified DNA concatemers on a glass surface;
  • FIG. 8D shows patterned amplified DNA concatemers on a PEG hydrogel surface (the region on the left is the surface of a naked PEG gel; the region on the right is decorated with DNA concatemers);
  • FIG. 9 shows two copies of T-junctions structures self-assembled from four red, blue, yellow, and violet 1 mm x 1 mm x 0.3 mm PEG hydrogel cuboids carrying patterned DNA concatemers (in the schematic, the parts that contain DNA are dark gray and the sequences are labeled with letters, where x and x* denote two complementary DNA sequences); and
  • FIG. 10A shows a non-limiting example of a fabrication schematic of cell-encapsulated hydrogel cubes.
  • FIGs. 10B- 10D show phase contrast (B, D) and fluorescent (C, E) microscopy images of a cell viability assay.
  • FIGs. 10F and 10G show phase-contrast (F) and fluorescent (G) microscopy images of cell-encapsulated hydrogel dimer assembled from human umbilical vein endothelial cell (HUVEC) (green) and smooth muscle cell (SMC) (red) encapsulated hydrogels.
  • UUVEC human umbilical vein endothelial cell
  • SMC smooth muscle cell
  • Tissue engineering holds great promise for developing therapies to treat injured tissue; however, existing technologies face several challenges.
  • the inability to mimic the complex microarchitecture of tissues limits the application of the scaffold. Provided herein are embodiments that address each of these challenges.
  • nucleic acid hydrogel subunits can be conjugated to biocompatible, shaped hydrogel subunits to form "self-assembling" units, referred to herein as "nucleic acid hydrogel subunits.”
  • the shaped hydrogel subunits (which may be referred to simply as “hydrogel subunits") provided herein are modified with (e.g., attached to) nucleic acids that can hybridize to each other through complementary (or sequence-specific) nucleic acid hybridization.
  • the nucleic acids attached to the hydrogel subunits function as "glue” to assemble the subunits, which assembly can be directed by introducing specific "address” sequences into the nucleic acids.
  • it is possible to produce a multitude of specific, stable, three-dimensional hydrogel structures e.g., linear chains and net-like structures) for use in many biomedical and bioengineering applications.
  • a basic self-assembly process using three nucleic acid hydrogel subunits is as follows: a first hydrogel subunit in the shape of a cube is conjugated on one side to a concatemer of sequence A, while the opposite side of the cube is conjugated to a concatemer of sequence B.
  • the A sequence of the concatemer of the first subunit will hybridize to another concatemer of sequence A* (complementary to A) conjugated on one side of a second cube.
  • the concatemer of sequence B of the first subunit will hybridize to its complementary sequence on a third shaped nucleic acid hydrogel subunit designed to have a concatemer of sequence complementary to B. In this way, a basic "chain" of three subunits is formed.
  • This process can continue, using multiple hydrogel subunits of various shapes, each conjugated to at least one (one or more) specifically designed nucleic acid concatemers, to create chains, sheets or other intricate architectures, depending on the shapes of the hydrogel subunits and how they are attached to each other.
  • the nucleic acid hydrogel subunits can be used in combination with existing tissue and cell (e.g., stem cell) bioengineering technology to create micro- vascularized tissue architectures for use in applications such as, for example, skin grafting to support cell growth and function.
  • tissue and cell e.g., stem cell
  • cells or other biomolecules may be embedded in the nucleic acid hydrogel subunits for delivery in vivo to a site of interest such as, for example, an injured tissue to promote tissue cell growth and vascularization.
  • the nucleic acid hydrogel subunits may be produced using a biocompatible gel such as polyethylene glycol (PEG) and may be seeded with cells such as, but not limited to, stem cells, progenitor cells or differentiated cells, depending on the application, and then used to produce tissue scaffolds (e.g., in vivo grafts). Cells may be embedded into the hydrogel subunits during subunit formation. In some instances, however, cells may be embedded into the hydrogel subunits after subunit formation.
  • the hydrogel subunits are conjugated to at least one nucleic acid designed to have a specific address sequence, which will be amplified to produce a concatemer of that sequence. Such addressable subunits can be directed to assemble into a two- or three-dimensional structure, mimicking an in vivo tissue structure.
  • Hydrogel subunits of the invention may be synthesized by any means known to one of ordinary skill in the art including, without limitation, photolithography, emulsification, microfluidic synthesis and micromolding (see, e.g., Khademhosseini and Lander, Biomaterials, 28:5087-5092 (2007); Khademhosseini et al, Proc. Natl. Acad. Sci. USA, 103:2480-2487 (2006), each of which is incorporated herein by reference).
  • photolithography may be used for a variety of biomedical applications to engineer hydrogel subunits, including microscale subunits.
  • synthetic or natural photocrosslinkable prepolymers may be crosslinked to form hydrogel subunits (Peppas et al., Adv. Mater., 18: 1-17 (2006), incorporated herein by reference).
  • hydrogel subunits Peppas et al., Adv. Mater., 18: 1-17 (2006), incorporated herein by reference.
  • UV ultraviolet
  • photolithography may be used to create the shaped hydrogel subunits or to immobilize cells within the shaped hydrogel subunits.
  • the photocrosslinkable hydrogel subunits of the invention may be made from various types of synthetic polymers including, without limitation, polyethylene glycol (PEG) (Koh et al., Langmuir, 18(7):2459-62 (2002); Liu et al., Biomed. Microdev., 4(4):257-66 (2002), each of which is incorporated herein by reference).
  • PEG polyethylene glycol
  • natural photocrosslinkable prepolymers may be used to form the hydrogel subunits (Khademhosseini et al. , J Biomed Mater Res A (2006), incorporated herein by reference).
  • photolithography may be used to conjugate chemical entities (e.g., nucleic acids) to hydrogel subunits with controlled spatial resolution (Hahn, et al., Adv. Mater. 18(20):2679-84 (2006); Luo et al., Nat Mater., 3(4):249- 53 (2004), each of which is incorporated herein by reference).
  • chemical entities e.g., nucleic acids
  • hydrogel subunits with controlled spatial resolution
  • emulsification may be used to fabricate hydrogel subunits of the invention.
  • a multi-phase mixture is stirred to generate small aqueous droplets of hydrogel precursors within an organic phase.
  • the size of the droplets may be controlled by the degree of mechanical agitation, viscosity of each phase, or the presence of surfactants that can modify the surface tension between the two phases.
  • the resulting droplets may be gelled using a variety of crosslinking mechanisms to generate spherical hydrogels.
  • cells may be added to the aqueous phase to fabricate cell-encapsulated, or cell- embedded, hydrogel subunits.
  • microfluidics may be used to fabricate nucleic acid hydrogel subunits of the invention.
  • a multi-phase system may be used to generate microparticles.
  • the viscous and surface tension forces may be used to create homogeneous particles that can be crosslinked to form microscale hydrogel subunits.
  • Such crosslinking may be, without limitation, chemical crosslinking or pH crosslinking.
  • a range of particle sizes and shapes may be created based on the design of the microfluidic channels. For example, by changing the dimensions of the microchannels, the flow rates and the droplet shapes, it is possible to create hydrogels in the form of spheres and rods (Xu et ah, Angew. Chem. Int. Ed. Engl., 44(25):3799 (2005), incorporated herein by reference).
  • microfluidic fabrication may be used to control the spatial properties of hydrogel subunits.
  • a microfluidic device that creates concentration gradients at two or more inlets may be used to create hydrogels with controlled gradients of signaling molecules or material properties embedded in the hydrogel subunit (Burdick et al. , Langmuir, 20(13):5153-6 (2004), incorporated herein by reference).
  • such hydrogel subunits may be used for various tissue-engineering applications in which concentration gradients are desired in the scaffolds.
  • Janus particles i.e., particles with two or more distinct sides
  • micromolding may be used to generate hydrogel subunits. Precursor polymers may be initially molded and subsequently gelled to generate structures of a variety of shapes and sizes (Fukada et al., Biomaterials, 27: 1479-1486 (2006); Yeh et al. Biomaterials, 27:5391-5398 (2006); Ling et al, Lab Chip, 7:756-762; Franzesi et al, J Am Chem Soc; 128(47): 15064-5 (2006), each of which is incorporated herein by reference). In some embodiments, micromolding may be used to generate three-dimensional hydrogel structures or microstructures. This may be accomplished by first using a sacrificial template around which the hydrogel can be formed (Stachowiak et al., Adv. Mater., 17(4):399-403 (2005), incorporated herein by reference).
  • biocompatible gel composition refers to any non-toxic, aqueous-based, biodegradable composition including, without limitation, polyethylene glycol (PEG), polyethylene glycol diacrylate (PEGDA), gelatin, agarose, collagen, calcium alginate, hyaluronic acid, hydrophobically modified chitosan, fibrogen and polysaccharides (Becker et al., Neurosurgery, 56(4):793-801 (2005); Himeda et al., Journal of Gynecological Surgery, 20(2):39-46 (2004); Dennis et al, Soft Matter, 7(9):4170-73 (2011), Khademhosseini et al, Lab Chip, 4(5):425-30 (2004); U.S. Patent No. 7,709,462, each of which is incorporated herein by reference).
  • Other biocompatible gel compositions may be used in accordance with the invention.
  • hydrogel subunit formation follows. A 2.0 percent by weight (wt %) sodium alginate prepolymer solution, containing biotin-conjugated alginate and streptavidin- conjugated DNA primers, is applied to a microfabricated stencil that is reversibly sealed to a flat substrate. Alginate gels modified with DNA primers are produced (immersion in 100 mM CaCi 2 , 30-90 min, room temperature) and stabilized (in 0.9 wt % NaCl, 5.0 M sodium triphosphate, 30 minutes, room temperature). Finally, concatemers are amplified from the
  • Divalent cation ⁇ e.g., Ca 2+ ) induced gelation of alginate is reversed by ethylenediaminetetraacetic acid (EDTA) or citric acid treatment, and such controlled dissolution is used to control lumen formation.
  • EDTA ethylenediaminetetraacetic acid
  • citric acid treatment ethylenediaminetetraacetic acid
  • the hydrogel subunits described herein may be a range of subunit sizes and shapes including, without limitation, hemi- sphere, cube, cuboidal, tetrahedron, cylinder, cone, octahedron, prism, sphere, pyramid, dodecahedron, tubular, irregular or abstract. Other hydrogel subunit sizes and shapes may be used in accordance with the invention.
  • the hydrogel subunits may vary in diameter, or edge length, from about 1 micrometer ( ⁇ ) to about 1 millimeter (mm). In some embodiments, the subunits are about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 100 ⁇ , or about 1 ⁇ to about 500 ⁇ in diameter (or edge length).
  • the hydrogel subunits are about 10 ⁇ , about 20 ⁇ , about 30 ⁇ , about 40 ⁇ , about 50 ⁇ , about 60 ⁇ , about 70 ⁇ , about 80 ⁇ , about 90 ⁇ , about 100 ⁇ , about 150 ⁇ , about 200 ⁇ , about 250 ⁇ , about 300 ⁇ , about 350 ⁇ , about 400 ⁇ , about 450 ⁇ m, about 500 ⁇ , about 550 ⁇ , about 600 ⁇ m, about 650 ⁇ , about 700 ⁇ m, about 750 ⁇ , about 800 ⁇ , about 850 ⁇ m, about 900 ⁇ , about 950 ⁇ m or about 1000 ⁇ in diameter (or edge length).
  • the hydrogel subunits are submicron scale, while in other embodiments, the subunits are as large as 1 centimeter (cm).
  • the "edge length" of a hydrogel subunit refers to the length of one edge of the hydrogel subunit such as, for example, a cube (having a total of 12 edges).
  • the nucleic acid hydrogel subunits of the invention comprise, on at least one of their surfaces, a nucleic acid.
  • the nucleic acids attached to the hydrogel subunits are referred to herein as being surface accessible, to denote their presence at the surface of the subunit and their ability to interact with other nucleic acids, including those on other hydrogel subunits.
  • nucleic acid refers to a polymeric form of
  • a nucleic may comprise naturally occurring nucleotides and/or non-naturally occurring nucleotides such as modified nucleotides, for example, methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before, or after, the nucleic acid is conjugated to the hydrogel subunit.
  • nucleic acids include, without limitation, single- stranded or partially single- stranded DNA, double- stranded or partially double- stranded DNA, single- stranded or partially single- stranded RNA, double- stranded or partially double- stranded RNA, cDNA, aptamers, peptide nucleic acids ("PNA"), 2'-5' DNA (a synthetic material with a shortened backbone that has a base-spacing that matches the A conformation of DNA - 2'-5' DNA will not normally hybridize with DNA in the B form, but it will hybridize readily with RNA), and locked nucleic acids ("LNA").
  • PNA peptide nucleic acids
  • Nucleic acid analogues include known analogues of natural nucleotides that have similar or improved binding, hybridization or base- pairing properties.
  • "Analogous" forms of purines and pyrimidines are well known in the art and include, without limitation, aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannos
  • DNA backbone analogues include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, morpholino carbamate, peptide nucleic acids (PNAs), methylphosphonate linkages, and alternating methylphosphonate and phosphodiester linkages.
  • PNAs peptide nucleic acids
  • a nucleic acid of invention may be further modified, such as by conjugation with a labeling component. Other nucleic acid modifications may be used.
  • a nucleic acid of the invention may be linear or circular.
  • a nucleic acid of the invention may be recombinant or isolated.
  • nucleic acid refers to a nucleic acid of genomic origin, cDNA origin, semi- synthetic origin or synthetic origin, which either does not occur in nature or is linked to another nucleic acid in a non-natural arrangement.
  • isolated nucleic acid refers to a nucleic acid of natural or synthetic origin, or some combination thereof, which (1) is separated, at least in part, from the environment and/or components with which it exists, and/or (2) is operably linked to a nucleic acid to which it is not linked in nature.
  • the nucleic acids of the invention may be extracted from cells or synthetically prepared according to any means known to those skilled in the art. For example, the nucleic acids may be chemically synthesized, transcribed or reverse transcribed from cDNA, transcribed or reverse transcribed from mRNA, or transcribed or reverse transcribed from other sources.
  • nucleic acids As used herein, two nucleic acids, or two nucleic acid regions or sequences, are two nucleic acids, or two nucleic acid regions or sequences.
  • Regions of complementarity between nucleic acids may range in length from about 5 to about 100 nucleotides. For example, regions of complementarity between nucleic acids may be about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides in length. In some embodiments, regions of complementarity between nucleic acids may be more than 100 nucleotides in length. In some embodiments, a nucleic acid may be about 15 nucleotides to about 1000 nucleotides in length.
  • the nucleic acid may be about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 or about 1000 nucleotides in length.
  • nucleic acid “primer,” as used herein, refers to a short nucleic acid (e.g., less than
  • a nucleic acid primer is about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20 or about 15 nucleotides in length.
  • Nucleic acids may be conjugated (or linked, as the terms are used interchangeably herein) to the hydrogel subunits using any technique known in the art.
  • N-hydroxysuccinimide (NHS) chemistry is used to conjugate the nucleic acids to the hydrogel subunits (see FIG. 2).
  • amine-bearing nucleic acids may be conjugated to PEG- NHS monomers using a standard protocol, as described in further detail in the Examples section.
  • other chemical crosslinking agents are used.
  • Examples of chemical crosslinking agents for use in accordance with the invention include, without limitation, thiol-thiol, amine-amine, amine-thiol and amine-acrylate, any of which may be used to conjugate DNA with, for example, PEG or other polypeptide hydrogel polymers.
  • biotin-modified DNA may be linked to a hydrogel subunit modified with tethered strep tavidin.
  • a shaped hydrogel subunit is conjugated to a nucleic acid, and then the nucleic acid is amplified to produce a concatemer.
  • a nucleic acid (e.g., DNA) concatemer is produced through rolling circle amplification (RCA) (Schopf et al., Anal Biochem 397: 115-117 (2011), incorporated herein by reference) or a variation thereof (Fire and Xu, Proc. Natl. Acad. Sci.
  • Rolling circle amplification involves two simultaneous processes. DNA polymerase synthesizes sequences complementary to a circular template, and as this replication proceeds, the parental duplex is unwound to allow the polymerase to advance. Continued DNA synthesis produces multiple single- stranded linear copies of the original DNA in a continuous head-to-tail series, forming a concatemer.
  • a DNA concatemer may also be referred to as a "giant DNA” or a "DNA nanoball,” as concatemers tend to coil into a large, ball-like shape.
  • Concatemers of the invention may comprise about 2 to about 1000 copies of the same nucleotides sequence.
  • concatemers may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 copies of the same nucleotides sequence.
  • concatemers may comprise more than 1000 copies of the same nucleotide sequence.
  • a stencil-based photolithography is used to pattern three distinct concatemer species on specific surfaces of polyethylene glycol (PEG) and gelatin gels, as illustrated in FIG. 1.
  • PEG polyethylene glycol
  • step (1) a stencil with 100 ⁇ size cubic holes is produced using standard techniques.
  • step (2) a hydrogel precursor solution (PEG-diacrylate (DA) or gelatin methacrylate), including photo- initiator and PEG-acrylate conjugated DNA primer 1, is applied to the stencil, which is sandwiched between two hydrophobic glass slides (pre-treated with octadecyltrichlorosilane). The shaped hydrogels form upon UV-crosslinking, and subsequently the glass slides are detached.
  • DA polyethylene glycol
  • step (2) a hydrogel precursor solution (PEG-diacrylate (DA) or gelatin methacrylate), including photo- initiator and PEG-acrylate conjugated DNA primer 1
  • DA polyethylene glycol
  • step (2) a hydrogel precursor solution (PEG-di
  • step (3) a prepolymer hydrogel solution containing gelatin methacrylate, or PEG- DA and PEG-acrylate, conjugated to DNA primer 2 is applied on the top surface, which is then covered with one glass slide.
  • a photomask is aligned with the shaped hydrogel subunit in stencil over the glass slide for secondary ultra violet (UV)-crosslinking of DNA primer 2 to the top surface of the hydrogel subunits.
  • UV ultra violet
  • the bottom surface is modified with DNA primer 3.
  • step (4) the stencil is peeled off, and hydrogel subunits with primer 2 on top, primer 3 on bottom, and primer 1 on the sides are collected.
  • step (5) the primers are converted into concatemers (DNA concatemers) by using rolling circle amplification (RCA) in aqueous solution with circular DNA templates complementary to primers 1, 2, and 3, respectively.
  • RCA rolling circle amplification
  • the gel is treated with three DNA probes, respectively complementary to primers 1, 2 and 3. Each probe is labeled with a distinct fluorophore. Three-color multiplexed imaging is used to quantify each primer density. Similarly, multiplexed imaging with complementary fluorescent DNA probes is used to quantify concatemer density. Additionally, concatemers are selectively released from the gel through endonuclease treatment and quantified by gel electrophoresis.
  • biocompatible shaped hydrogels e.g., resembling a natural extracellular matrix
  • the shaped hydrogel subunits of the invention may be linked to form a single chain or a sheet of multiple chains linked together (e.g., similar to a net or net-like structure).
  • the term "linked” refers to the joining of at least two shaped hydrogel subunits through their nucleic acid-modified surfaces.
  • subunit A is considered to be linked to subunit B if a nucleic acid conjugated to subunit A hybridizes, in a sequence- specific manner, to a nucleic acid conjugated to subunit B.
  • the shaped subunits are directed to form simple chains, sheets, or more complex branched structures (e.g., T-junctions).
  • each shaped hydrogel subunit may be designed to have particular nucleic acids conjugated to specific surfaces, or specific surface regions, such that nucleic acid hybridization of complementary sequences between neighboring nucleic acids brings together the hydrogel subunits in a predictable manner.
  • the shaped hydrogel subunits are linked such that the individual subunits form a larger, more intricate shape, for example, mimicking a particular tissue architecture such as, for example, branched vasculature.
  • nucleic acid hydrogel subunits (e.g., within a mixed population of complementary nucleic acid hydrogel subunits) self-assemble under conditions that allow hybridization of sequences having at least 65% complementarity.
  • the nucleic acid hydrogel subunits self-assemble with a sequence specificity of at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98%.
  • non-specific binding between nucleic acid hydrogel subunits is less than 20%.
  • non-specific binding between nucleic acid hydrogel subunits is less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5%.
  • Nucleic acid hydrogel subunits of the invention can be assembled in an aqueous assembly system or an interfacial assembly system.
  • An aqueous assembly system refers to a system in which hydrogels are suspended in solution and when rotated can freely move in all directions.
  • an aqueous assembly system may be comprised of a microtube filled with phosphate buffered saline (PBS) supplemented with 0.5 M NaCl, 0.5mM EDTA and 0.05% TWEEN ® 20.
  • PBS phosphate buffered saline
  • An interfacial assembly system refers to a system in which hydrogels are floated on an interface formed between aqueous buffer and hydrophobic liquid (e.g., aqueous PBS and liquid fluor-inert FC-40) and under mild shaking conditions can only move along the liquid/liquid interface.
  • aqueous buffer and hydrophobic liquid e.g., aqueous PBS and liquid fluor-inert FC-40
  • the nucleic acid hydrogel subunits of the invention may be used to generate structures (e.g., microstructures) of defined and predetermined shape and complexity. Such structures may be used as tissue grafts in vivo or in vitro.
  • the hydrogel subunits may be seeded with (e.g., encapsulated with) living cells such as stem cells, progenitor cells and/or differentiated cells.
  • the stem cells may be embryonic stem cells or adult stem cells. Examples of stem cells for use in accordance with the invention include, without limitation, endothelial stem cells, mesenchymal stem cells and hematopoietic stem cells.
  • progenitor cells for use in accordance with the invention include, without limitation, angioblasts and endothelial progenitor cells.
  • differentiated cells for use in accordance with the invention include, without limitation, red blood cells, white blood cells, platelets, stromal cells, fat cells, bone cells, skin cells and muscle cells.
  • the cells are fibroblasts.
  • the cells are induced pluripotent cells.
  • the hydrogel subunits encapsulate biomolecules such as, for example, proteins, lipids, polysaccharides, metabolites, nucleic acids or any combination of two or more of the foregoing.
  • nucleic acid hydrogel structures of the invention may be used as, or as part of, a tissue graft.
  • tissues that may be grafted using the nucleic acid hydrogel structures of the invention include, without limitation, skin, bone, nerves, tendons, neurons, blood vessels, fat and cornea.
  • a hydrogel subunit-based skin graft may be used, for example, to treat skin loss due to a wound, burn, infection or surgery.
  • a hydrogel subunit- based vascular graft may be used, for example, as prosthetic blood vessels in surgical procedures.
  • kits comprising the shaped hydrogel subunits of the invention with attached non-amplified primers or attached concatemers.
  • the invention also provides kits for the synthesis of the shaped hydrogel subunits, including subunits with non-amplified primers or subunits with the amplified concatemers. Examples
  • a strategy was developed to use complementary DNA molecules as "glue” to direct the self-assembly of hydrogel cubes (also referred to herein as subunits) with edge lengths of 250 ⁇ .
  • step (1) amine-bearing short DNA strands (brown, 56 nt) were conjugated to PEG-NHS monomers (MW 3500 Da) using a standard protocol (Schlingman et al., B: Biointerfaces 83: 91-95 (2011), incorporated herein by reference).
  • step (2) the DNA-PEG-acrylate was mixed with photocrosslinkable poly(ethylene glycol)- diacrylate (PEG-DA, 4000 MW) and 0.5 wt % photoinitiator, and exposed to UV under a photomask with 250 ⁇ x 250 ⁇ square holes.
  • the height of the cubes was controlled by using microscope cover glass slides (No. 2; 250 um in thickness) as spacers.
  • 250 ⁇ x 250 ⁇ x 250 ⁇ hydrogel cubes uniformly modified with short DNA primers, were produced.
  • step (3) the DNA primers hybridized with complementary, circular DNA templates (produced by circularization of short linear DNA using
  • CIRCLIGASETM CIRCLIGASETM
  • RCA rolling circle amplification
  • the DNA primers were amplified to produce long strands with repeated sequences complementary to the circular template, referred to herein as concatemers, DNA concatemers or "giant" DNAs.
  • DNA concatemer-directed assembly of hydrogel cubes was next demonstrated. Using the procedure described above, 250 ⁇ x 250 ⁇ x 250 ⁇ hydrogel cubes carrying DNA concatemers containing tandem repeated complementary 48-nucleotide sequences (FIG. 3A) were fabricated. The DNA concatemers were uniformly amplified on the surface of hydrogel cubes, with one cube having on its surface an "a" sequence and the other cube having on its surface an "a*" complementary sequence (such sequences are shown in FIG. 3A as SEQ ID NO: l (top) and SEQ ID NO:42 (bottom)).
  • Hydrogel cubes carrying complementary DNA "a” or "a*” were labeled with red or blue fluorescent microbeads, respectively, and stained with SYBR® Gold. Hybridization between the complementary DNA sequences resulted in assembly of hydrogel cubes. Self-assembly was performed by mixing these hydrogel cubes in a 0.5 ml microtube filled with assembly buffer under mild rotation, using a tube rotator with a fixed speed of 18 rpm (FIG. 3B, see details under Method heading below).
  • Including DNA-free yellow hydrogel cubes (i.e., cubes that contain yellow microbeads) in the reaction system did not change the assembly outcome for either the hydrogel cubes carrying 56-nt short DNA primers (FIG. 3E, left) or hydrogel cubes carrying DNA concatemers (FIG. 3E, right). Moreover, yellow hydrogel cubes were not observed in the assembled structure, confirming that the assembly was directed by DNA concatemers on the hydrogel cube surface.
  • concatemers was capable of directing assembly of hydrogel cubes with a wide range of edge lengths. Cubes carrying complementary DNA "a” or "a*” were labeled with red or blue color microbeads respectively. The assembly reaction was performed as described above.
  • FIG. 3H shows a schematic for multiplexed self-assembly of 25 orthogonal pairs of dimers in five independent experiments.
  • the assembly process was conducted using agitation of repeated mild rotation at a fixed speed of 18 rpm and strong hand shaking every 30 minutes to disrupt non-specific binding.
  • the final assembled nucleic acid hydrogel structures from the five independent experiments were pooled together into a single Petri dish for imaging and quantification.
  • Each of the 25 expected specific hydrogel dimers were all identified (FIGs. 31, 3J; the assembled structures are highlighted by white triangles; scale bar: 1 mm).
  • FIG. 3 J shows a schematic (top left corner) depicting the double-component structure of the hydrogel cube used in the multiplexed self-assembly of 25 hydrogel cube dimers.
  • the core "pad” cube was 100 ⁇ x 100 ⁇ x 100 ⁇
  • the periphery "body” cube was 300 ⁇ x 300 ⁇ x 300 ⁇ .
  • the core and periphery hydrogel cubes were labeled with distinct, colored microbeads, and pairwise combinations of five colored cubes (red, blue, yellow, black and violet) generated 25 distinct signatures.
  • Example 2 Self-assembly ofhydrogel cubes with face-specific DNA modifications.
  • hydrogel cubes were fabricated with face-specific DNA concatemer modification.
  • the procedure is illustrated in FIG. 4.
  • steps 1-4 a procedure is described for making a two-component cube composite structure where a larger "body-cube” displays smaller DNA-modified "pad-cubes" on its designated sides; in steps 5 and 6, the agitation system for their assembly is described.
  • Step 1 The 150 ⁇ x 150 ⁇ x 150 ⁇ hydrogel pad cubes were made from a precursor solution that contained 20 wt % PEGDA (4 KDa) and PEG (3.5 KDa) acrylate single- stranded DNA primers using photolithography. A photomask with 150 ⁇ x 150 ⁇ square holes was used to control the cross-section shape of the pad cube. Microscope cover glass slides (No. 1; 150 ⁇ in thickness) were used as spacers to control the height of the pad cube.
  • Step 2 The un-polymerized reagent was washed away, and DNA concatemers were produced through a RCA reaction, as described above.
  • FIG. 4A shows arrays of 150 um pad cubes (dark gray) with uniform DNA concatemer modification.
  • Step 3 To make the larger body cube, a second solution containing only 20 wt % polyethylene glycol diacrylate (PEGDA) (4 KDa) was added, and the cube was covered with a second photomask with 250 ⁇ x 250 ⁇ square holes. This photomask was aligned carefully with the pad cubes made in step 2 such that this photomask covered half of the cross-section area of each pad cubes (to protect them from subsequent UV exposure). Microscope cover glass slides (No. 2; 250 ⁇ in thickness) were used as spacers to control the height of the body cubes.
  • PEGDA polyethylene glycol diacrylate
  • Step 4 Subsequent ultraviolet UV treatment resulted in the polymerization of the second 250 ⁇ x 250 ⁇ x 250 ⁇ body cube. Un-polymerized reagent was washed away.
  • an array of hydrogel cubes was produced: the 250 ⁇ body cube covered half of the 150 ⁇ pad cubes— only the 150 ⁇ pad cubes were modified with DNA concatemers.
  • the hydrogel cube composite had only DNA concatemer modification on designated faces that display the pads.
  • This composite structure may be referred to herein as a hydrogel cube with surface- specific DNA modifications.
  • Step 5 The hydrogel cubes were collected into a 0.5 ml microtube filled with the assembly buffer.
  • Step 6 Assembly was performed by rotating the tube.
  • Step 7 The solution was transferred to a Petri dish and imaged under a microscope. Using the above strategy, the multiplexed assembly of three hydrogel cube dimer species was demonstrated (FIGs. 4B and 4C). In this experiment, six hydrogel cube species were manufactured (FIG. 4B, left). The first species was a red hydrogel cube ⁇ i.e..
  • FIG. 4B depicts hydrogel cubes displaying face- specific DNA concatemers with tandem repeats and assembled cubes based on "a/a*", “b/b*”, or "c/c*”complementarity.
  • the smaller pad cube carrying the DNA concatemer is depicted in dark gray.
  • hydrogel cubes that display DNA concatemers on multiple designated faces
  • linear chain structures and net-like structures were next constructed.
  • two hydrogel cube species were made: a red cube that displays DNA concatemer "a” on two opposite faces, and a blue cube that displays DNA concatemer "a*” on two opposite faces (FIG. 4D, left, schematic).
  • the assembly of these two species produced chain structures that contained alternating red and blue hydrogel cubes, as expected (FIG. 4D, right, microscopy image).
  • the longest chain observed contained seven cubes (FIG. 4D, top-right corner).
  • hydrogel cubes were then made: (i) a red cube species that displays DNA concatemer "a” on two opposite faces and DNA concatemer "b” on another two opposite faces, and (ii) a blue cube species that displays pairs of "a*" and "b*” on opposite faces (FIG. 4E, left).
  • the assembly of these two species resulted in the formation of net-like structures with alternating red and blue hydrogel cubes that were connected through DNA modified sides (FIG. 4E, right).
  • the linear and net-like structures were each assembled in three independent experiments, where each experiment used 40 red and 40 blue complementary cubes.
  • Example 3 Interfacial self-assembly of hydrogel cuboids into complex structures.
  • hydrogel cuboids were next directed to assemble into prescribed, finite structures.
  • hydrogel cuboids were fabricated and floated on a liquid/liquid interface between aqueous phosphate buffered saline (PBS) and fluor-inert FC-40 liquid, and horizontal shaking was applied to facilitated assembly (FIG. 5A).
  • PBS phosphate buffered saline
  • FC-40 fluor-inert FC-40 liquid
  • a two-component hydrogel composite was made: the body cuboids were 1 mm (length) x 1 mm (width) x 0.3 mm (height), and the pads were DNA-modified 250 ⁇ x 250 ⁇ x 250 ⁇ cubes. Note that the length ratio between the body cuboid and the pad cubes was increased to 4: 1 (compare to the 2.5: 1 in FIG. 4).
  • the interfacial system and the cuboids that carry relatively smaller pad cubes it was possible to assemble dimers (FIG. 5B), linear chains with finite length (FIG. 5C), a T-junction (FIG. 5D) and a square structure (FIG. 5E).
  • the linear chain, the T-junction and the square were all composed of four distinct cuboid species.
  • the assembled hydrogel structure was changed from a chain to a T-junction to a square.
  • the self-assembly of two T- junctions in the same reaction system was also demonstrated.
  • FIGs. 6A and 6B illustrate another example of assembling the shaped nucleic acid hydrogels.
  • Two hydrogel cubes were first aligned with respect to the center of each cube.
  • a small area on top surface of a hydrogel cube was modified with DNA. This was achieved by aligning a secondary mask having a specific design of 150 ⁇ in diameter with the center of the hydrogel cube having a diameter of about 250 ⁇ , followed by concatemerization of the DNA.
  • a dimeric structure was assembled from the hydrogel cubes with patterned DNA on the top surface.
  • the alignment of assembled hydrogel cubes was controlled through patterning DNA on different areas of the hydrogel surface ⁇ e.g., at the corner or at the center of the cube) (FIGs. 6D and 6E), and the orientation of hydrogel in the final assembled structure was controlled through patterning the DNA on the hydrogel surface with a cross design (FIGs. 6B and 6C).
  • Example 5 Cell encapsulated hydrogel fabrication
  • NIH 3T3 cells were encapsulated inside PEG hydrogel cubes, and DNA amplification was performed in a cell medium-based reaction solution. As showed in FIG. 7, amplified DNA was detected on hydrogel cubes with cells encapsulated therein. Fluorescent imaging of SYBR ® Green stained concatemers in cell-encapsulated and cell-free hydrogel cubes (control) were compared to quantify the effects of the cell cultures on RCA efficiency. The efficiency was subsequently optimized by varying RCA reaction conditions as well as the density and size of DNA concatemers. Similarly, imaging of the change of fluorescence after various incubation time (-1-24 hours) after RCA was used to study the stability/degradation of DNA concatemers in cell-encapsulated hydrogels. The hydrogels were imaged after saline (e.g., PBS) washing to remove potential DNA degradation products.
  • saline e.g., PBS
  • FIG. 10A shows a fabrication schematic of cell-encapsulated hydrogel cubes in accordance with the invention.
  • a DNA pad-cube was fabricated as described in FIG. 2A.
  • a DNA concatemer was amplified from the tethered ssDNA primer using rolling circle amplification (RCA).
  • RCA rolling circle amplification
  • a cell-encapsulated body-cube was fabricated from a polymer precursor solution containing living cells. The final micro structure was controlled by aligning the second photomask with the first small pad-cube as described in FIG. 4A and in the Examples.
  • FIGs. 10B-10D show phase contrast (FIGs. 10B, 10D) and fluorescent (FIGs.
  • FIGs. 10F and 10G show phase-contrast (FIG. 10F) and fluorescent (FIG. 10G) microscopy images of cell-encapsulated hydrogel dimer assembled from human umbilical vein endothelial cell (HUVEC) and smooth muscle cell (SMC) encapsulated hydrogels.
  • the invention provides DNA-directed self-assembly of shape-controlled hydrogel subunits to build complex structures in a programmable fashion. Acting like sequence specific glue and tethered onto a hydrogel surface, single- stranded DNA concatemers exhibit a significant capability for binding objects across scales, with sizes ranging from 30 micrometers to a millimeter (or more). Additionally, DNA concatemers offer significant diversity over current mesoscale self-assembly systems: 50 DNA sequences were designed to generate 25 orthogonal pairs of specific interactions. The designable DNA "glues" thus provide improved methods for programming (e.g., mesoscale) self-assembly.
  • hydrogel subunit fabrication is important.
  • a precisely controlled fabrication technique by which specific DNA concatemers are decorated on a prescribed face of a hydrogel subunit (e.g., cube).
  • a hydrogel subunit e.g., cube
  • hydrogel cuboids can be fabricated with four different DNA concatemers on four designated faces, and by simply changing the surface DNA decoration pattern, discrete hydrogel structures were assembled, including dimers, T-junctions, linear chains with fixed length and squares.
  • Another aspect of the invention is biocompatibility.
  • Preliminary experiments suggest that mammalian cells encapsulated inside hydrogel subunits maintained high viability through the fabrication and assembly process (FIGs. 10A-G).
  • the DNA-directed hydrogel self- assembly system provided herein has promising potential in bottom-up tissue engineering applications.
  • Cell-laden hydrogel units may be engineered to self-assemble into structures that mimic the microarchitecture of native tissues.
  • DNA-directed self-assembly of shape-controlled hydrogel subunits proved to be highly programmable and controllable and will open new doors to address the challenge of building complex self-assembled 3D structures for diverse applications.
  • the prepolymer solution of PEG-DA with average molecular weight 4000 Da was prepared by diluting PEG (Monomer- Polymer&Dajac Labs) in DPBS (Gibco) to a final concentration of 20 wt % with 1 wt % photo-initiator, l-(4-(2-Hydroxyethoxy)- phenyl)- 2-hydroxy-2-methyl-l -propane- 1 -one (IRGACURE 2959 Ciba) for hydrogel modules fabrication.
  • Circular DNA template was produced by ligating 5' and 3' terminal of
  • oligonucleotides (DNA) (Invitrogen) with 5 '-terminal phosphate modification using a
  • DNA sequence for DNA glue synthesis was designed using software of nucleic acid package (NUPACK) with minimized mis-hybridization between each two non-complementary sequences and ordered from Invitrogen.
  • NUPACK nucleic acid package
  • Baseline-ZEROTM DNase was obtained from Epicentre Biotechnology and used with final concentration of 1 U/ml.
  • Sequence design Twenty-five orthogonal sequence pairs were designated using a modified version of the Domain Design (DD) software described by Zhang et al. (Zhang et al., Lecture Notes in Computer Science 6518: 162-175 (2011), incorporated herein by reference). Twenty-five domains of 24 bases each were first designed, and then these domains were concatenated together into 25 domains of 48 bases each. Sequences were designed using a three-letter alphabet to reduce spurious hybridization. In order to reduce long regions of repeated bases (e.g., poly-A, poly-G, etc.), sequences, which had a higher Shannon entropy, were rewarded.
  • DD Domain Design
  • the NUPACK thermodynamic analysis package was used to calculate that unintended interactions between the concatenated products would be -108 times less favorable than the intended interactions (Dirks et ah, SIAM Rev 49:65-88 (2007), incorporated herein by reference).
  • oligonucleotides DNA primer with a amine modification at the 5 '-terminal of Poly(T-36) linker to the acrylate-PEG-NHS (Jenkem Technology).
  • Shape-controlled PEG hydrogel was fabricated by following a general photolithograph process, as described (Du et ah, Proc. Natl. Acad. Sci. USA 105: 9522-9527 (2008), incorporated herein by reference), in which photomask was designed using AutoCAD software with 20,0000 dpi resolution (CAD/ Art Services;
  • DNA concatemers were amplified by soaking the hydrogel subunits in reaction solution including 50 ⁇ circular DNA template and 5 U/ ⁇ of Phi29 DNA polymerase (EPICENTRE Biotechnology) in lx DNA amplification buffer at 37 °C overnight, according the manufacturer's instructions.
  • Hydrogel subunits carrying patterned DNA concatemers were fabricated in a two-step fabrication process.
  • DNA concatemers were amplified on a small hydrogel cube with size of 150- 300 ⁇ , fabricated by photolithography, as described above.
  • the DNA modified hydrogel subunits were washed thoroughly with lx assembly buffer (0.5 M NaCl, 0.5 mM EDTA, and 0.05% Tween-20 in lx general PBS buffer), and then a 20 wt % prepolymer PEG solution, including 1 v/v% color microbeads, was added.
  • the final shape of the hydrogel subunits was controlled by a secondary photomask being aligned with the DNA hydrogel subunits in accordance with the designed patterning of DNA under microscope.
  • Hydrogel subunits carrying specific DNA concatemers were collected in a 0.5 ml microtube fulfilled with assembly buffer containing 0.5 M NaCl, 0.5 mM EDTA, and 0.05% Tween-20 in lx general PBS buffer.
  • assembly buffer containing 0.5 M NaCl, 0.5 mM EDTA, and 0.05% Tween-20 in lx general PBS buffer.
  • the inside surface of microtube was treated with a corona treater (BD-20AC from Electro-Technic Products Inc.) and coated with 10% PEGDA (MW 1000) beforehand.
  • the microtube was subjected to agitation of continuous 360-degree upright rotation on a VWR Multimix tube rotator with a fixed speed of 18 rpm and intermittent soft vortex or hand shaking every 30-60 minutes to disrupt non-specific binding or aggregates.
  • the hydrogel subunits were transferred to a petri dish filled with solution of 20 wt % PEG (MW 3350) in lx assembly buffer and subject to further horizontal shaking with speed of about 60 rpm on a VWR standard orbital shaker.
  • Assembled hydrogel structure were identified, quantified and imaged using a Stereo Microscope.
  • Cell-laden hydrogel subunits were assembled in a general culture medium, Dulbecco' s Modified Eagle's medium (DMEM, Invitrogen), without serum supplement.
  • DMEM Dulbecco' s Modified Eagle's medium
  • Liquid/liquid interface self-assembly.
  • Liquid/liquid interface was generated between Fluorinert electronic liquid FC-40 (bottom liquid, 3MTM Chemicals) and aqueous assembly buffer (top liquid) in a Petri dish.
  • Hydrogel subunits were floated on the interface and subjected to agitation of continuous horizontal shaking at low speed of 60 rpm on a VWR standard orbital shaker (Model 1000, VWR) and intermittent 120 rpm shaking or strong hand shaking every 30-60 minutes to break undesired, spurious aggregates.
  • the assembly process was recorded using an image recording software, HyperCam Version 2, under a Stereo Microscope.
  • RCA DNA product was amplified and analyzed on a 1% agarose gel.
  • Surface of hydrogel carrying giant DNA glue was analyzed using a scanning electron microscopy (Zeiess EVO SEM).
  • Hydrogel carrying ssDNA probe was fabricated on a glass slide surface as described earlier and giant single- strand DNA was amplified by soaking hydrogel in RCA reaction solution including 50 ⁇ circular DNA template and 5 U/ ⁇ of Phi29 DNA polymerase (EPICENTRE Biotechnology) in lx DNA amplification buffer at 37 °C overnight according to the manufacturer's instructions.
  • the hydrogel was rinsed thoroughly with general PBS (GIBCO, DPBS), and liquid around the gel was dried carefully using Kimwipes. Following washing, the hydrogel was frozen in -80 °C freezer for 3 hours and transferred to a freeze drier for 2 days for scanning electron microscopy imaging.
  • D025, D025* and poly(T-36) with a 5'-terminal amine modification were ordered from Integrated DNA Technology (IDT) and dissolved in water upon arrival.
  • IDT Integrated DNA Technology
  • One phosphate group was added to the 3'- terminal of DNA oligos by T4 Polynucleotide kinase (PNK, EPICENTRE Biotechnology) for ligation with poly(T- 36) or circularization.
  • PNK Polynucleotide kinase
  • Modification master reaction comprising of 33 mM Tris-HCl (pH 7.5), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, 5 mM ATP, 100 ⁇ DNA oligo and 10 U/ ⁇ PNK enzyme, was incubate in 37 °C for 3 hours and then PNK enzyme was inactivated by incubation at 70 °C for 30min.
  • Circular DNA template was prepared by circulating phosphate modified DNA oligo using Cirligase II ssDNA ligase (EPICENTRE Biotechnology).
  • Circularization reaction was performed with supplementing 2.5 mM manganese chloride, 1 M Betaine, and 5 U/ ⁇ ligase in phosphate modified DNA oligo solution and incubate at 60 °C for 6 hours, and ligase was inactivated at 80 °C for 10 minutes. Following this, exonuclease I (EPICENTRE
  • DNA primer was prepared by ligating modified DNA oligos to amine- poly(T-36). Ligation reaction was performed with supplementing 20 U/ ⁇ T4 RNA ligase (New England Biolab) and 100 ⁇ amine-poly(T-36) in phosphate modified DNA oligo solution and incubated at room temperature (25 °C) overnight.
  • Cubic PEG hydrogel subunits with a size of 250 ⁇ x 250 ⁇ x 250 ⁇ carrying DNA primer was fabricated and giant DNA was amplified as described earlier.
  • pairwise combination of 5 color microbeads including polybead Red Dyed 1.0 ⁇ microspheres, polybead Blue Dyed 0.5 ⁇ microspheres, Polybead Yellow Dyed 3.0 ⁇ microspheres, polybead Violet Dyed 1.0 ⁇ microsphere and polybead Black Dyed 10.0 ⁇ microspheres (Polysciences) generated 25 distinct labeling.
  • the fabrication process is similar to that of hydrogel for dimer assembly, as described herein, but with minor modification.
  • the second hydrogel (periphery part) was fabricated to completely warp the first hydrogel (core part) forming the final structure.
  • a single copy of each hydrogel uniformly carrying DNA D001, D001*, D002, D002* D025, D025* was collected in 1.5 ml Eppendorf microtubes fulfilled with lx assembly buffer and self-assembly was performed in aqueous liquid as described above.
  • NIH-3T3 mouse fibroblast cells, smooth muscle cells (SMCs), and GFP- transfected human umbilical vein endothelial cells (HUVECs) expressing green florescence protein were cultured using an approved protocol.
  • SMC's were cultured in SMC basal medium (RPIM 1640; Invitrogen; Carlsbad, CA).
  • NIH-3T3 fibroblast cells were encapsulated inside hydrogels and cell viability was analyzed after assembly. Cells were trypsinized, counted, and re-suspended inside the prepolymer solution at concentration of 1x107 cells/ml. After fabrication, cells containing hydrogels were placed in PCR Eppendorf tubes (0.5 ml; Hamburg, Germany) filled with cell culture medium and rotated for 3 hours. Cell-encapsulated hydrogels were transferred into a Petri dish and the viability of the cells was determined using LIVE/DEAD Viability/Cytotoxicity Kit (2 ⁇ of Calcein AM and 0.5 ⁇ of Ethidium homodimer-1 in DPBS; Invitrogen; Carlsbad, CA).
  • HUVECs and SMCs were trypsinized, harvested, and labeled with PKH26 Red Florescent Cell Linker Kit (Sigma Aldrich; St. Louise, MO) and CellTrackerTM Green CMFDA (Invitrogen; Carlsbad, CA) accordingly.
  • PKH26 Red Florescent Cell Linker Kit Sigma Aldrich; St. Louise, MO
  • CellTrackerTM Green CMFDA Invitrogen; Carlsbad, CA
  • the nuclei of both groups of cells were stained with DAPI Nucleic Acid Stain (Invitrogen; Carlsbad, CA) to facilitate imaging.
  • Labeled cells were re-suspended in prepolymer solution (PEG 20 % wt) at a density of 1x107 cells/ml.
  • Single side-modified hydrogels were made out of the prepolymer-cell solution using the same method mentioned earlier.
  • Hydrogels were collected and placed in an Eppendorf tube containing a mixture of HUVEC and SMC cell culture medium (Dulbecco's Modified Eagle's medium, (DEME), Invitrogen) without serum and were rotated for 3 hours before being imaged.
  • DEME Dulbecco's Modified Eagle's medium
  • inventive embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.
  • inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein.
  • a reference to "A and/or B", when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • the phrase "at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.
  • At least one of A and B can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

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Abstract

La présente invention concerne des sous-motifs d'hydrogel d'acides nucléiques mis en forme par auto-assemblage et des procédés pour les utiliser.
PCT/US2013/070260 2012-11-16 2013-11-15 Auto-assemblage d'hydrogel d'acides nucléiques Ceased WO2014078636A1 (fr)

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WO2021211873A1 (fr) * 2020-04-15 2021-10-21 The Penn State Research Foundation Conjugué adn-alginate biodégradable pour marquage et imagerie réversibles de protéines et de cellules
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