Gradually Self-Strengthen DNA Supramolecular Hydrogels.

The dynamic mechanical strength of the extracellular matrix (ECM) has been demonstrated to play important role in determining the cell behavior. Growing evidences suggest that the gradual stiffening process of the matrix is particularly decisive during tissue development and wound healing. Herein, a novel strategy to prepare hydrogels with gradually enhanced mechanical strength is provided. Such hydrogels could maintain the dynamic properties at their initial states, such as self-healing and shear-thinning properties. With subsequent slow covalent crosslinking, the stability and mechanical properties would be gradually improved. This method is useful for sequence programmability and oxidation strategies, which has provided an alternated tool to study cell behavior during dynamic increase in mechanical strength of ECM.

Advanced Smart Biomaterials for Regenerative Medicine and Drug Delivery Based on Phosphoramidite Chemistry: From Oligonucleotides to Precision Polymers.

Over decades of development, while phosphoramidite chemistry has been known as the leading method in commercial synthesis of oligonucleotides, it has also revolutionized the fabrication of sequence-defined polymers (SDPs), offering novel functional materials in polymer science and clinical medicine. This review has introduced the evolution of phosphoramidite chemistry, emphasizing its development from the synthesis of oligonucleotides to the creation of universal SDPs, which have unlocked the potential for designing programmable smart biomaterials with applications in diverse areas including data storage, regenerative medicine and drug delivery. The key methodologies, functions, biomedical applications, and future challenges in SDPs, have also been summarized in this review, underscoring the significance of breakthroughs in precisely synthesized materials.

Rational Design of DNA Hydrogels Based on Molecular Dynamics of Polymers.

In recent years, DNA has emerged as a fascinating building material to engineer hydrogel due to its excellent programmability, which has gained considerable attention in biomedical applications. Understanding the structure-property relationship and underlying molecular determinants of DNA hydrogel is essential to precisely tailor its macroscopic properties at molecular level. In this review, the rational design principles of DNA molecular networks based on molecular dynamics of polymers on the temporal scale, which can be engineered via the backbone rigidity and crosslinking kinetics, are highlighted. By elucidating the underlying molecular mechanisms and theories, it is aimed to provide a comprehensive overview of how the tunable DNA backbone rigidity and the crosslinking kinetics lead to desirable macroscopic properties of DNA hydrogels, including mechanical properties, diffusive permeability, swelling behaviors, and dynamic features. Furthermore, it is also discussed how the tunable macroscopic properties make DNA hydrogels promising candidates for biomedical applications, such as cell culture, tissue engineering, bio-sensing, and drug delivery.

Preparing liposomes through frame guided assembly with high-loading functional nucleic acids.

Recently, a frame guided assembly (FGA) has been demonstrated as a robust tool to prepare liposomes with customized morphologies. However, the potential application of FGA liposomes in delivering nucleic acid drugs is still limited by the low payload. In this study, by systemically investigating the correlation between the leading hydrophobic group (LHG) density and the initial detergent concentration, it has been demonstrated that frames with a low LHG density, which may facilitate the increase of the load of the nucleic acid drug, could be guided to form liposomes at low initial detergent concentrations. By capitalizing on this phenomenon, FGA liposomes with a high loading of ASO/siRNA have been successfully prepared and applied to treat pathogenic genes.

Increase Nebulization Stability of Lipid Nanoparticles by Integrating a DNA Supramolecular Hydrogel.

Nebulized lipid nanoparticles (LNPs) have been considered as potential therapies for genetic disease as well as infectious disease. However, the sensitivity of LNPs to high shear stress during the nebulization process results in loss of the integrity of the nanostructure and the capability of delivering active pharmaceutical ingredients. Herein we have provided a fast extrusion method to prepare liposomes incorporated with a DNA hydrogel (hydrogel-LNPs) to improve the stability of the LNPs. Taking advantage of the good cellular uptake efficiency, we also demonstrated the potential of hydrogel-LNPs in delivering small molecular doxorubicin (Dox) and nucleic acid drugs. This work provides not only highly biocompatible hydrogel-LNPs for aerosol delivery, but also a strategy to regulate the elasticity of LNPs, which will benefit the potential optimization of drug delivery carriers.

DNA Materials Assembled from One DNA Strand.

Due to the specific base-pairing recognition, clear nanostructure, programmable sequence and responsiveness of the DNA molecule, DNA materials have attracted extensive attention and been widely used in controlled release, drug delivery and tissue engineering. Generally, the strategies for preparing DNA materials are based on the assembly of multiple DNA strands. The construction of DNA materials using only one DNA strand can not only save time and cost, but also avoid defects in final assemblies generated by the inaccuracy of DNA ratios, which potentially promote the large-scale production and practical application of DNA materials. In order to use one DNA strand to form assemblies, the sequences have to be palindromes with lengths that need to be controlled carefully. In this review, we introduced the development of DNA assembly and mainly summarized current reported materials formed by one DNA strand. We also discussed the principle for the construction of DNA materials using one DNA strand.

Effects of Univariate Stiffness and Degradation of DNA Hydrogels on the Transcriptomics of Neural Progenitor Cells.

Mechanical interactions between cells and extracellular matrix (ECM) are critical for stem cell fate decision. Synthetic models of ECM, such as hydrogels, can be used to precisely manipulate the mechanical properties of the cell niche and investigate how mechanical signals regulate the cell behavior. However, it has long been a great challenge to tune solely the ECM-mimic hydrogels' mechanical signals since altering the mechanical properties of most materials is usually accompanied by chemical and topological changes. Here, we employ DNA and its enantiomers to prepare a series of hydrogels with univariate stiffness regulation, which enables a precise interpretation of the fate decision of neural progenitor cells (NPCs) in a three-dimensional environment. Using single-cell RNA sequencing techniques, Monocle pseudotime trajectory and CellphoneDB analysis, we demonstrate that the stiffness of the hydrogel alone does not influence the differentiation of NPCs, but the degradation of the hydrogel that enhances cell-cell interactions is possibly the main reason. We also find that ECM remodeling facilitates cells to sense mechanical stimuli.

Single-Molecular Poly(propylene oxide) (PPO) Nucleus-Guided Assembly for Hydrophobicity-Dependent Molecular Transport in the Nanopore.

By combining DNA nanotechnology and solid-phase nanopore technology, the aggregation behavior of polymer guided by a single-molecular poly(propylene) (PPO) nucleus in a 3D DNA network has been studied. At low temperature, the PPO chain is evenly dispersed in the rigid 3D DNA network; at higher temperature, the PPO chain self-collapses to a single-molecular nucleus; and upon addition of amphiphilic block copolymers below the critical micelle concentration (CMC), the chains tend to aggregate on the isolated hydrophobic nucleus through intermolecular hydrophobic interactions. The process has been characterized by a rheological test and an electrochemical test. This study not only provides a preliminary understanding of the nucleation and growth process of block copolymers but also offers a theoretical basis for the study of protein self-folding and aggregation in the future. On this basis, utilizing this nucleation and growth event, a novel smart nanopore has been developed for hydrophobicity-dependent molecular transport.

Reinforcing supramolecular hyaluronan hydrogels via kinetically interlocking multiple-units strategy.

Supramolecular hydrogels exhibit promising potential in biological and clinical fields due to their special dynamic properties. However, most existing supramolecular hydrogels suffer from poor mechanical strength, which severely limits their applications. Here in this study, the Kinetically Interlocking Multiple-Units (KIMU) strategy was applied to the hyaluronan networks by introducing different supramolecular interaction motifs in an organized and alternative manner. Our strategy successfully elevated the energy barrier of crosslinker dissociation to 103.0 kJ mol-1 and increased the storage modulus of hydrogels by 78 % with the intrinsic dynamic properties preserved. It can be expected that this method would bring a convenient and effective route to fabricate novel supramolecular materials with excellent mechanical properties.

A DNA-Schiff base functional nanopore sensing platform for the highly sensitive detection of Al3+ and Zn2+ ions.

The combination of DNA nanotechnology and nanopore sensing technology has greatly promoted research on target molecule or ion detection. The large solid-state nanopores/nanochannels show better mechanical stability and reproducibility, but metal ion detection in the large nanopores with diameters of hundreds of nanometers or several micrometers is rarely reported. Hence, it is meaningful and urgent to develop a large nanopore-based sensing platform for the detection of metal ions. Herein, we employed a salicylic aldehyde-modified DNA network in conjunction with a glass nanopipette (GN) with a diameter of hundreds of nanometers as a sensing platform for the detection of target metal ions. Upon the addition of different receptors with the amino group, the salicylic aldehyde could in situ specifically recognize and bind with Zn2+ and Al3, forming Schiff base-metal ion complexes at the four vertices of one face per nanocube unit. The steric hindrance effect of multiple Schiff bases and metal ion complexes leads to the blockage of internal structure and decrease of ion current in the GN. Owing to this signal amplification strategy, the detection limit of the target metal ion reaches a level of fM in the GN with a diameter of about 300 nm. In the future, this functional nanopore sensing platform is expected to realize highly sensitive detection for more biological metal ions by choosing appropriate receptors.

Studies on the Synergistic Effect of Tandem Semi-Stable Complementary Domains on Sequence-Defined DNA Block Copolymers.

Tandem semi-stable complementary domains play an important role in life, while the role of these domains in the folding process of nucleic acid molecules has not been systematically studied. Here, we designed a clean model system by synthesizing sequence-defined DNA-OEG copolymers composed of ssDNA fragments with palindromic sequences and orthogonal oligo(tetraethylene glycol) (OEG) linkers. By altering the lengths of DNA units (6-12 nt) and OEG linkers (Xn = 0-4) separately, we systematically studied how stabilities of tandem complementary domains and connecting flexibilities affect the assembly topology. Combining experimental methods and coarse-grained molecular simulation analysis, distributions of multiple assembled conformations (mainly monomers, dimers, and clusters) were characterized. Both results indicated that tandem semi-stable complementary domains tend to form homogeneous closed circular dimers instead of larger clusters due to the synergistic enhancement effect, and the distributions of each conformation highly depend on flexibilities.

"Shutter" Effects Enhance Protein Diffusion in Dynamic and Rigid Molecular Networks.

Hydrogels have been widely applied to understand the fundamental functions and mechanism of a natural extracellular matrix (ECM). However, revealing the high permeability of ECM through synthetic hydrogels is still challenged by constructing analogue networks with rigid and dynamic properties. Here, in this study, taking advantage of the rigidity and dynamic binding of DNA building blocks, we have designed a model hydrogel system with structural similarity to ECM, leading to enhanced diffusion for proteins compared with a synthetic polyacrylamide (PAAm) hydrogel. The molecular diffusion behaviors in such a rigid and dynamic network have been investigated both in experiments and simulations, and the dependence of diffusion coefficients with respect to molecular size exhibits a unique transition from a power law to an exponential function. A "shutter" model based on the rigid and dynamic molecular network has been proposed, which has successfully revealed how the rigidity and dynamic bond exchange determine the diffusion mechanism, potentially providing a novel perspective to understand the possible mechanism of enhanced diffusion behaviors in ECM.

Frame-Guided Assembly of Amphiphiles.

Amphiphiles tend to self-assemble into various structures and morphologies in aqueous environments (e.g., micelles, tubes, fibers, vesicles, and lamellae). These assemblies and their properties have made significant impact in traditional chemical industries, e.g., increasing solubility, decreasing surface tension, facilitating foaming, etc. It is well-known that the molecular structure and its environment play a critical role in the assembly process, and many theories, including critical packing factor, thermodynamic models, etc., have been proposed to explain and predict the assembly morphology. It has been recognized that the morphology of the amphiphilic assembly plays important roles in determining the functions, such as curvature-dependent biophysical (e.g., liposome fusion and fission) and biochemical (e.g., lipid metabolism and membrane protein trafficking) processes, size-related EPR (enhanced permeability and retention) effects, etc. Meanwhile, various nanomaterials have promised great potential in directing the arrangement of molecules, thus generating unique functions. Therefore, control over the amphiphilic morphology is of great interest to scientists, especially in nanoscale with the assistance of functional nanomaterials. However, how to precisely manipulate the sizes and shapes of the assemblies is challenged by the entropic nature of the hydrophobic interaction. Inspired by the "cytoskeleton-membrane protein-lipid bilayer" principle of the cell membrane, a strategy termed "frame-guided assembly (FGA)" has been proposed and developed to direct the arrangement of amphiphiles. The FGA strategy welcomes various nanomaterials with precisely controlled properties to serve as scaffolds. By introducing scattered hydrophobic molecules, which are defined as either leading hydrophobic groups (LHGs) or nucleation seeds onto a selected scaffold, a discontinuous hydrophobic trace along the scaffold can be outlined, which will further guide the amphiphiles in the system to grow and form customized two- or three-dimensional (2D/3D) membrane geometries.Topologically, the supporting frame can be classified as three types including inner-frame, outer-frame, and planar-frame. Each type of FGA assembly possesses particular advantages: (1) The inner-frame, similar to endoskeletons of many cellular structures, steadily supports the membrane from the inside and exposes the full surface area outside. (2) The outer-frame, on the other hand, molds and constrains the membrane-wrapped vesicles to regulate their size and shape. It also allows postengineering of the frame to precisely decorate and dynamically manipulate the membrane. (3) The planar-frame mediates the growth of the 2D membrane that profits from the scanning-probe microscopic characterization and benefits the investigation of membrane proteins.In this Account, we introduce the recent progress of frame-guided assembly strategy in the preparation of customized amphiphile assemblies, evaluate their achievements and limitations, and discuss prospective developments and applications. The basic principle of FGA is discussed, and the morphology controllability is summarized in the inner-, outer-, and planar-frame categories. As a versatile strategy, FGA is able to guide different types of amphiphiles by designing specific LHGs for given molecular structures. The mechanism of FGA is then discussed systematically, including the driving force of the assembly, density and distribution of the LHGs, amphiphile concentration, and the kinetic process. Furthermore, the applications of FGA have been developed for liposome engineering, membrane protein incorporation, and drug delivery, which suggest the huge potential of FGA in fabricating novel and functional complexes.

DNA-Modified Liquid Crystal Droplets.

In this work, we have combined the advantages of sequence programmability of DNA nanotechnology and optical birefringence of liquid crystals (LCs). Herein, DNA amphiphiles were adsorbed onto LC droplets. A unique phenomenon of LC droplet aggregation was demonstrated, using DNA-modified LC droplets, through complementary DNA hybridization. Further functionalization of DNA-modified LC droplets with a desired DNA sequence was used to detect a wide range of chemicals and biomolecules, such as Hg2+, thrombin, and enzymes, through LC droplet aggregation and vice versa, which can be seen through the naked eye. These DNA-modified LC droplets can be printed onto a desired patterned surface with temperature-induced responsiveness and reversibility. Overall, our work is the first to report DNA-modified LC droplet, which provides a general detection platform based on the development of DNA aptamers. Additionally, this work inspires the exploration of surface information visualization combined with microcontact printing.

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.

Genetically Encoded Double-Stranded DNA-Based Nanostructure Folded by a Covalently Bivalent CRISPR/dCas System.

DNA nanotechnology has been widely employed in the construction of various functional nanostructures. However, most DNA nanostructures rely on hybridization between multiple single-stranded DNAs. Herein, we report a general strategy for the construction of a double-stranded DNA-ribonucleoprotein (RNP) hybrid nanostructure by folding double-stranded DNA with a covalently bivalent clustered regularly interspaced short palindromic repeats (CRISPR)/nuclease-dead CRISPR-associated protein (dCas) system. In our design, dCas9 and dCas12a can be efficiently fused together through a flexible and stimuli-responsive peptide linker. After activation by guide RNAs, the covalently bivalent dCas9-12a RNPs (staples) can precisely recognize their target sequences in the double-stranded DNA scaffold and pull them together to construct a series of double-stranded DNA-RNP hybrid nanostructures. The genetically encoded hybrid nanostructure can protect genetic information in the folded state, similar to the natural DNA-protein hybrids present in chromosomes, and elicit efficient stimuli-responsive gene transcription in the unfolded form. This rationally developed double-stranded DNA folding and unfolding strategy presents a new avenue for the development of DNA nanotechnology.

Anti-Friction MSCs Delivery System Improves the Therapy for Severe Osteoarthritis.

Osteoarthritis (OA) is a musculoskeletal disorder disease affecting about 500 million people worldwide and mesenchymal sem cells (MSCs) therapy has been demonstrated as a potential strategy to treat OA. However, the shear forces during direct injection and the harsher shear condition of OA environments would lead to significant cell damage and inhibit the therapeutic efficacy. Herein, DNA supramolecular hydrogel has been applied as delivering material for MSCs to treat severe OA model, which perform extraordinary protection in MSCs against the shear force both in vitro and in vivo. It is demonstrated that the DNA supramolecular hydrogel can promote formation of quality cartilage, reduce osteophyte, and normalize subchondral bone under the high friction condition of OA, whose molecular mechanisms underlying therapeutic effects are also investigated. It can be anticipated that DNA supramolecular hydrogel would be a promising cell delivery system for multiple potential MSCs therapy.

Shear-Thinning and Designable Responsive Supramolecular DNA Hydrogels Based on Chemically Branched DNA.

A novel supramolecular DNA hydrogel system was designed based on a directly synthesized chemically branched DNA. For the hydrogel formation, a self-dimer DNA with two sticky ends was designed as the linker to induce the gelation of B-Y. By programing the linker sequence, thermal and metal-ion responsiveness could be introduced into this hydrogel system. This supramolecular DNA hydrogel shows shear-thinning, designable responsiveness, and good biocompatibility, which will simplify the hydrogel composition and preparation process of the supramolecular DNA hydrogel and accelerate its biomedical applications.

Highly Permeable DNA Supramolecular Hydrogel Promotes Neurogenesis and Functional Recovery after Completely Transected Spinal Cord Injury.

Regeneration after severe spinal cord injury cannot occur naturally in mammals. Transplanting stem cells to the injury site is a highly promising method, but it faces many challenges because it relies heavily on the microenvironment provided by both the lesion site and delivery material. Although mechanical properties, biocompatibility, and biodegradability of delivery materials have been extensively explored, their permeability has rarely been recognized. Here, a DNA hydrogel is designed with extremely high permeability to repair a 2 mm spinal cord gap in Sprague-Dawley rats. The rats recover basic hindlimb function with detectable motor-evoked potentials, and a renascent neural network is formed via the proliferation and differentiation of both implanted and endogenous stem cells. The signal at the lesion area is conveyed by, on average, 15 newly formed synapses. This hydrogel system offers great potential in clinical trials. Further, it should be easily adaptable to other tissue regeneration applications.