DNA hydrogels for bone regeneration.

DNA-based biomaterials have been proposed for tissue engineering approaches due to their predictable assembly into complex morphologies and ease of functionalization. For bone tissue regeneration, the ability to bind Ca2+ and promote hydroxyapatite (HAP) growth along the DNA backbone combined with their degradation and release of extracellular phosphate, a known promoter of osteogenic differentiation, make DNA-based biomaterials unlike other currently used materials. However, their use as biodegradable scaffolds for bone repair remains scarce. Here, we describe the design and synthesis of DNA hydrogels, gels composed of DNA that swell in water, their interactions in vitro with the osteogenic cell lines MC3T3-E1 and mouse calvarial osteoblast, and their promotion of new bone formation in rat calvarial wounds. We found that DNA hydrogels can be readily synthesized at room temperature, and they promote HAP growth in vitro, as characterized by Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and transmission electron microscopy. Osteogenic cells remain viable when seeded on DNA hydrogels in vitro, as characterized by fluorescence microscopy. In vivo, DNA hydrogels promote the formation of new bone in rat calvarial critical size defects, as characterized by micro-computed tomography and histology. This study uses DNA hydrogels as a potential therapeutic biomaterial for regenerating lost bone.

Quantitative Characterization of RCA-Based DNA Hydrogels - Towards Rational Materials Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively. Primer concentration and stabilizing additives showed minimal impact on RCA efficiency, while changes in polymerase and nucleotide concentrations had a stronger effect. Concentration of the circular template exerted the greatest influence on RCA productivity. An exponential correlation between hydrogel viscosity and DNA amplicon concentration was observed, with nucleobase sequence significantly affecting both amplification efficiency and material properties, particularly through secondary structures. This study suggests that combining high-throughput experimental methods with structural folding prediction offers a viable approach for systematically establishing structure-property relationships, aiding the rational design of DNA hydrogel material systems.

DNA hydrogels and their derivatives in biomedical engineering applications.

Deoxyribonucleotide (DNA) is uniquely programmable and biocompatible, and exhibits unique appeal as a biomaterial as it can be precisely designed and programmed to construct arbitrary shapes. DNA hydrogels are polymer networks comprising cross-linked DNA strands. As DNA hydrogels present programmability, biocompatibility, and stimulus responsiveness, they are extensively explored in the field of biomedicine. In this study, we provide an overview of recent advancements in DNA hydrogel technology. We outline the different design philosophies and methods of DNA hydrogel preparation, discuss its special physicochemical characteristics, and highlight the various uses of DNA hydrogels in biomedical domains, such as drug delivery, biosensing, tissue engineering, and cell culture. Finally, we discuss the current difficulties facing DNA hydrogels and their potential future development.

A Critical View on the Use of DNA Hydrogels in Cell-Free Protein Synthesis.

Numerous studies have reported in the past that the use of protein-encoding DNA hydrogels as templates for cell-free protein synthesis (CFPS) leads to better yields than the use of conventional templates such as plasmids or PCR fragments. Systematic investigation of different types of bulk materials from pure DNA hydrogels and DNA hydrogel composites using a commercially available CFPS kit showed no evidence of improved expression efficiency. However, protein-coding DNA hydrogels were advantageously used in microfluidic reactors as immobilized templates for repetitive protein production, suggesting that DNA-based materials offer potential for future developments in high-throughput profiling or rapid in situ characterization of proteins.

pH-Responsive and Reversible A-Motif-Based DNA Hydrogel: Synthesis and Biosensing Application.

Functional DNA hydrogels with various motifs and functional groups require perfect sequence design to avoid cross-bonding interference with themselves or other structural sequences. This work reports an A-motif functional DNA hydrogel that does not require any sequence design. A-motif DNA is a noncanonical parallel DNA duplex structure containing homopolymeric deoxyadenosines (poly-dA) strands that undergo conformation changes from single strands at neutral pH to a parallel duplex DNA helix at acidic pH. Despite this and other advantages over other DNA motifs like no cross-bonding interference with other structural sequences, the A-motif has not been explored much. We successfully synthesized a DNA hydrogel by using an A-motif as a reversible handle to polymerize a DNA three-way junction. The A-motif hydrogel was initially characterized by electrophoretic mobility shift assay, and dynamic light scattering, which showed the formation of higher-order structures. Further, we used imaging techniques like atomic force microscopy and scanning electron microscope to validating its hydrogel like highly branched morphology. pH-induced conformation transformation from monomers to gel is quick and reversible, and was analysed for multiple acid-base cycles. The sol-to-gel transitions and gelation properties were further examined in rheological studies. The use of the A-motif hydrogel in the visual detection of pathogenic target nucleic acid sequence was demonstrated for the first time in a capillary assay. Moreover, pH-induced hydrogel formation was observed in situ as a layer over the mammalian cells. The proposed A-motif DNA scaffold has enormous potential in designing stimuli-responsive nanostructures that can be used for many biological applications.

Programmable DNA Hydrogels as Artificial Extracellular Matrix.

The cell microenvironment plays a crucial role in regulating cell behavior and fate in physiological and pathological processes. As the fundamental component of the cell microenvironment, extracellular matrix (ECM) typically possesses complex ordered structures and provides essential physical and chemical cues to the cells. Hydrogels have attracted much attention in recapitulating the ECM. Compared to natural and synthetic polymer hydrogels, DNA hydrogels have unique programmable capability, which endows the material precise structural customization and tunable properties. This review focuses on recent advances in programmable DNA hydrogels as artificial extracellular matrix, particularly the pure DNA hydrogels. It introduces the classification, design, and assembly of DNA hydrogels, and then summarizes the state-of-the-art achievements in cell encapsulation, cell culture, and tissue engineering with DNA hydrogels. Ultimately, the challenges and prospects for cellular applications of DNA hydrogels are delivered.

Thermal-Responsive MXene-DNA Hydrogel for Near-Infrared Light Triggered Localized Photothermal-Chemo Synergistic Cancer Therapy.

Stimuli-responsive DNA hydrogels are promising candidates for cancer treatment, as they not only possess biocompatible and biodegradable 3D network structures as highly efficient carriers for therapeutic agents but also are capable of undergoing programmable gel-to-solution transition upon external stimuli to achieve controlled delivery. Herein, a promising platform for highly efficient photothermal-chemo synergistic cancer therapy is established by integrating DNA hydrogels with Ti3 C2 TX -based MXene as a photothermal agent and doxorubicin (DOX) as a loaded chemotherapeutic agent. Upon the irradiation of near-infrared light (NIR), temperature rise caused by photothermal MXene nanosheets triggers the reversible gel-to-solution transition of the DOX-loaded MXene-DNA hydrogel, during which the DNA duplex crosslinking structures unwind to release therapeutic agents for efficient localized cancer therapy. Removal of the NIR irradiation results in the re-formation of DNA duplex structures and the hydrogel matrix, and the recombination of free DOX and adaptive hydrogel transformations can also be achieved. As demonstrated by both in vitro and in vivo models, the MXene-DNA hydrogel system, with excellent biocompatibility and injectability, dynamically NIR-triggered drug delivery, and enhanced drug uptake under mild hyperthermia conditions, exhibits efficient localized cancer treatment with fewer side effects to the organisms.

DNA Nanotechnology-Based Supramolecular Assemblies for Targeted Biomedical Applications.

DNA is a polyanionic, hydrophilic, and natural biopolymer that offers properties such as biodegradability, biocompatibility, non-toxicity, and non-immunogenicity. These properties of DNA as an ideal biopolymer offer modern-day researchers' reasons to exploit these to form high-order supramolecular assemblies. These structures could range from simple to complex and provide various applications. Among them, supramolecular assemblies like DNA hydrogels (DNA-HG) and DNA dendrimers (DNA-DS) show massive growth potential in the areas of biomedical applications such as cell biology, medical stream, molecular biology, pharmacology, and healthcare product manufacturing. The application of both of these assemblies has seen enormous growth in recent years. In this focused review on DNA-based supramolecular assemblies like hydrogels and dendrimers, we present the principles of synthesis and characterization, key developments with examples and applications, and conclude with a brief perspective on challenges and future outlook for such devices and their subsequent applications.

DNA Hydrogels in the Perspective of Mechanical Properties.

Tailoring the mechanical properties has always been a key to the field of hydrogels in terms of different applications. Particularly, DNA hydrogels offer an unambiguous way to precisely tune the mechanical properties, largely on account of their programmable sequences, abundant responding toolbox, and various ligation approaches. In this review, DNA hydrogels from the perspective of mechanical properties, from a synthetic standpoint to different applications, are introduced. The relationship between the structure and their mechanical properties in DNA hydrogels and the methods of regulating the mechanical properties of DNA hydrogels are specifically summarized. Furthermore, several recent applications of DNA hydrogels in relation to their mechanical properties are discussed. Benefiting from the tunability and flexibility, rational design of mechanical properties in DNA hydrogels provided unheralded interest from fundamental science to extensive applications.

A Biostable l-DNA Hydrogel with Improved Stability for Biomedical Applications.

DNA hydrogels have attracted increasing attention owing to their excellent permeability and high mechanical strength, together with thixotropy, versatile programmability and good biocompatibility. However, the moderate biostability and immune stimulation of DNA have arisen as big concerns for future potential clinical applications. Herein, we report the self-assembly of a novel l-DNA hydrogel, which inherited the extraordinary physical properties of a d-DNA hydrogel. With the mirror-isomer deoxyribose, this hydrogel exhibited improved biostability, withstanding fetal bovine serum (FBS) for at least 1 month without evident decay of its mechanical properties. The low inflammatory response of the l-DNA hydrogel has been verified both in vitro and in vivo. Hence, this l-DNA hydrogel with outstanding biostability and biocompatibility can be anticipated to serve as an ideal 3D cell-culture matrix and implanted bio-scaffold for long-term biomedical applications.

Kinetically Interlocking Multiple-Units Polymerization of DNA Double Crossover and Its Application in Hydrogel Formation.

A novel kinetically interlocking multiple-units (KIMU) supramolecular polymerization system with DNA double crossover backbone is designed. The rigidity of DX endows the polymer with high molecular weight and stability. The observed concentration of the formed polymers is insensitive and stable under ultralow monomer concentration owing to the KIMU interactions, in which multiple noncovalent interactions are connected by the phosphodiester bonds. Furthermore, a pH-responsive DNA supramolecular hydrogel is constructed by introducing a half i-motif domain into the DNA monomer. The rigidity of DNA polymer endows the hydrogel with high mechanical strength and low gelation concentration. This study enriches the KIMU strategy and offers a simple but effective way to fabricate long and stable supramolecular polymers by balancing the reversibility and stability. It also shows great potentials to construct next generation of smart materials, such as DNA nanostructures, DNA motors, and DNA hydrogels.

Target DNA- and pH-responsive DNA hydrogel-based capillary assay for the optical detection of short SARS-CoV-2 cDNA.

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.

Smart Bilayer Polyacrylamide/DNA Hybrid Hydrogel Film Actuators Exhibiting Programmable Responsive and Reversible Macroscopic Shape Deformations.

As a crucial instinct for the survival of organisms, adaptive smart deformation has been well shown via profusely astounding examples within biological morphogenesis in nature, which inspired the construction of biomimetic shape-morphing materials with controlled actuating behaviors. Herein, the construction of nature-inspired bilayer hydrogel film actuators, composed of a polyacrylamide hydrogel passive layer and a polyacrylamide-DNA hybrid hydrogel active layer, which exhibited programmable stimuli-responsive and reversible macroscopic shape deformations directed by the sequence of DNA crosslinking units in the active layer, is reported. As a proof-of-concept, the introduction of DNA i-motif based crosslinking structures into the active layer, which can undergo pH-stimulated formation and dissociation of crosslinking between polymers and therefore change the crosslinking density of the active layer, lead to the redistribution of the internal stresses within the bilayer structure, and result in the pH-stimulated shape deformations. By programming the sequence of DNA units in the active layer, a Ag+ /Cysteamine-stimulated bilayer DNA hybrid hydrogel film actuator is further constructed and exhibits excellent actuation behaviors. Thanks to the micrometer-scale thickness of the films, these actuators exhibit a high degree of macroscopic and reversible shape deformations at high speed, which may find use in future smart biosensing and biomedical applications.

Construction of Organelle-Like Architecture by Dynamic DNA Assembly in Living Cells.

The design of controllable dynamic systems is vital for the construction of organelle-like architectures in living cells, but has proven difficult due to the lack of control over defined topological transformation of self-assembled structures. Herein, we report a DNA based dynamic assembly system that achieves lysosomal acidic microenvironment specifically inducing topological transformation from nanoparticles to organelle-like hydrogel architecture in living cells. Designer DNA nanoparticles are constructed from double-stranded DNA with cytosine-rich stick ends (C-monomer) and are internalized into cells through lysosomal pathway. The lysosomal acidic microenvironment can activate the assembly of DNA monomers, inducing transformation from nanoparticles to micro-sized organelle-like hydrogel which could further escape into cytoplasm. We show how the hydrogel regulates cellular behaviors: cytoskeleton is deformed, cell tentacles are significantly shortened, and cell migration is promoted.

How to Construct DNA Hydrogels for Environmental Applications: Advanced Water Treatment and Environmental Analysis.

With high binding affinity, porous structures, safety, green, programmability, etc., DNA hydrogels have gained increasing recognition in the environmental field, i.e., advanced treatment technology of water and analysis of specific pollutants. DNA hydrogels have been demonstrated as versatile potential adsorbents, immobilization carriers of bioactive molecules, catalysts, sensors, etc. Moreover, altering components or choosing appropriate functional DNA optimizes environment-oriented hydrogels. However, the lack of comprehensive information hinders the continued optimization. The principle used to fabricate the most suitable hydrogels in terms of the requirements is the focus of this Review. First, different fabrication strategies are introduced and the ideal characteristic for environmental applications is in focus. Subsequently, recent environmental applications and the development of diverse DNA hydrogels regarding their synthesis mechanism are summarized. Finally, the Review provides an insight into the remaining challenging and future perspectives in environmental applications.

Smart DNA Hydrogel Integrated Nanochannels with High Ion Flux and Adjustable Selective Ionic Transport.

Nanochannels based on smart DNA hydrogels as stimulus-responsive architecture are presented for the first time. In contrast to other responsive molecules existing in the nanochannel in monolayer configurations, the DNA hydrogels are three-dimensional networks with space negative charges, the ion flux and rectification ratio are significantly enhanced. Upon cyclic treatment with K+ ions and crown ether, the DNA hydrogel states could be reversibly switched between less stiff and stiff networks, providing the gating mechanism of the nanochannel. Based on the architecture of DNA hydrogels and pH stimulus, cation or anion transport direction could be precisely controlled and multiple gating features are achieved. Meanwhile, G-quadruplex DNA in the hydrogels might be replaced by other stimulus-responsive DNA molecules, peptides, or proteins, and thus this work opens a new route for improving the functionalities of nanochannel by intelligent hydrogels.

Clamped Hybridization Chain Reactions for the Self-Assembly of Patterned DNA Hydrogels.

DNA hydrogels hold great potential for biological and biomedical applications owing to their programmable nature and macroscopic sizes. However, most previous studies involve spontaneous and homogenous gelation procedures in solution, which often lack precise control. A clamped hybridization chain reaction (C-HCR)-based strategy has been developed to guide DNA self-assembly to form macroscopic hydrogels. Analogous to catalysts in chemical synthesis or seeds in crystal growth, we introduced DNA initiators to induce the gelation process, including crosslinked self-assembly and clamped hybridization in three dimensions with spatial and temporal control. The formed hydrogels show superior mechanical properties. The use of printed, surface-confined DNA initiators was also demonstrated for fabricating 2D hydrogel patterns without relying on external confinements. This simple method can be used to construct DNA hydrogels with defined geometry, composition, and order for various bioapplications.

Toward Applications for DNA Nanotechnology-More Bricks To Build With.

Another brick in the wall: DNA nanotechnology has come a long way since its initial beginnings. This would not be possible without the continued development of methods for DNA assembly and new uses for DNA as a material. This Special Issue highlights some of the newest building blocks for nanodevices based on DNA.

A writable polypeptide-DNA hydrogel with rationally designed multi-modification sites.

A polypeptide-DNA hydrogel is prepared by employing the "X"-shaped DNA assembling structure as crosslinker. The hydrogel can be modified with multifunctional components (here fluorescent molecules as a model) and possesses excellent self-healing and thixotropic properties, enabling the direct-writing of arbitrary 3D structures. This study provides a simple, universal strategy for the assembly of functionalized hydrogels.

Construction of three-dimensional DNA hydrogels from linear building blocks.

A three-dimensional DNA hydrogel was generated by self-assembly of short linear double-stranded DNA (dsDNA) building blocks equipped with sticky ends. The resulting DNA hydrogel is thermoresponsive and the length of the supramolecular dsDNA structures varies with temperature. The average diffusion coefficients of the supramolecular dsDNA structures formed by self-assembly were determined by diffusion-ordered NMR spectroscopy (DOSY NMR) for temperatures higher than 60 °C. Temperature-dependent rheological measurements revealed a gel point of 42±1 °C. Below this temperature, the resulting material behaved as a true gel of high viscosity with values for the storage modulus G' being significantly larger than that for the loss modulus G''. Frequency-dependent rheological measurements at 20 °C revealed a mesh size (ξ) of 15 nm. AFM analysis of the diluted hydrogel in the dry state showed densely packed structures of entangled chains, which are also expected to contain multiple interlocked rings and catenanes.