Nebulized Inhalation of Peptide-Modified DNA Origami To Alleviate Acute Lung Injury.

Acute lung injury (ALI) is a severe inflammatory lung disease, with high mortality rates. Early intervention by reactive oxygen species (ROS) scavengers could reduce ROS accumulation, break the inflammation expansion chain in alveolar macrophages (AMs), and avoid irreversible damage to alveolar epithelial and endothelial cells. Here, we reported cell-penetrating R9 peptide-modified triangular DNA origami nanostructures (tDONs-R9) as a novel nebulizable drug that could reach the deep alveolar regions and exhibit an enhanced uptake preference of macrophages. tDONs-R9 suppressed the expression of pro-inflammatory cytokines and drove polarization toward the anti-inflammatory M2 phenotype in macrophages. In the LPS-induced ALI mouse model, treatment with nebulized tDONs-R9 alleviated the overwhelming ROS, pro-inflammatory cytokines, and neutrophil infiltration in the lungs. Our study demonstrates that tDONs-R9 has the potential for ALI treatment, and the programmable DNA origami nanostructures provide a new drug delivery platform for pulmonary disease treatment with high delivery efficiency and biosecurity.

Self-Assembled DNA Composite-Engineered Mesenchymal Stem Cells for Improved Skin-Wound Repair.

The direct use of mesenchymal stem cells (MSCs) as therapeutics for skin injuries is a promising approach, yet it still faces several obstacles, including limited adhesion, retention, and engraftment of stem cells in the wound area, as well as impaired regenerative and healing functions. Here, DNA-based self-assembled composites are reported that can aid the adhesion of MSCs in skin wounds, enhance MSC viability, and accelerate wound closure and re-epithelialization. Rolling-circle amplification (RCA)-derived DNA flowers, equipped with multiple copies of cyclic Arg-Gly-Asp (cRGD) peptides and anti-von Willebrand factor (vWF) aptamers, act as robust scavengers of reactive oxygen species (ROS) and enable synergistic recognition and adhesion to stem cells and damaged vascular endothelial cells. These DNA structure-aided stem cells are retained at localized wound sites, maintain repair function, and promote angiogenesis and growth factor secretion. In both normal and diabetes-prone db/db mice models with excisional skin injuries, facile topical administration of DNA flower-MSCs elicits rapid blood vessel formation and enhances the sealing of the wound edges in a single dose. DNA composite-engineered stem cells warrant further exploration as a new strategy for the treatment of skin and tissue damage.

Chemically Conjugated Branched Staples for Super-DNA Origami.

DNA origami, comprising a long folded DNA scaffold and hundreds of linear DNA staple strands, has been developed to construct various sophisticated structures, smart devices, and drug delivery systems. However, the size and diversity of DNA origami are usually constrained by the length of DNA scaffolds themselves. Herein, we report a new paradigm of scaling up DNA origami assembly by introducing a novel branched staple concept. Owing to their covalent characteristics, the chemically conjugated branched DNA staples we describe here can be directly added to a typical DNA origami assembly system to obtain super-DNA origami with a predefined number of origami tiles in one pot. Compared with the traditional two-step coassembly system (yields <10%), a much greater yield (>80%) was achieved using this one-pot strategy. The diverse superhybrid DNA origami with the combination of different origami tiles can be also efficiently obtained by the hybrid branched staples. Furthermore, the branched staples can be successfully employed as the effective molecular glues to stabilize micrometer-scale, super-DNA origami arrays (e.g., 10 × 10 array of square origami) in high yields, paving the way to bridge the nanoscale precision of DNA origami with the micrometer-scale device engineering. This rationally developed assembly strategy for super-DNA origami based on chemically conjugated branched staples presents a new avenue for the development of multifunctional DNA origami-based materials.

DNA Origami: From Molecular Folding Art to Drug Delivery Technology.

DNA molecules that store genetic information in living creatures can be repurposed as building blocks to construct artificial architectures, ranging from the nanoscale to the microscale. The precise fabrication of self-assembled DNA nanomaterials and their various applications have greatly impacted nanoscience and nanotechnology. More specifically, the DNA origami technique has realized the assembly of various nanostructures featuring rationally predesigned geometries, precise addressability, and versatile programmability, as well as remarkable biocompatibility. These features have elevated DNA origami from academic interest to an emerging class of drug delivery platform for a wide range of diseases. In this minireview, the latest advances in the burgeoning field of DNA-origami-based innovative platforms for regulating biological functions and delivering versatile drugs are presented. Challenges regarding the novel drug vehicle's safety, stability, targeting strategy, and future clinical translation are also discussed.

Genetically Encoded DNA Origami for Gene Therapy In Vivo.

DNA origami has played an important role in various biomedical applications, including biosensing, bioimaging, and drug delivery. However, the function of the long DNA scaffold involved in DNA origami has yet to be fully exploited. Herein, we report a general strategy for the construction of a genetically encoded DNA origami by employing two complementary DNA strands of a functional gene as the DNA scaffold for gene therapy. In our design, the complementary sense and antisense strands can be directly folded into two DNA origami monomers by their corresponding staple strands. After hybridization, the assembled genetically encoded DNA origami with precisely organized lipids on the surface can function as the template for lipid growth. The lipid-coated and genetically encoded DNA origami can efficiently penetrate the cell membrane for successful gene expression. After decoration with the tumor-targeting group, the antitumor gene (p53) encoded DNA origami can elicit a pronounced upregulation of the p53 protein in tumor cells to achieve efficient tumor therapy. The targeting group-modified, lipid-coated, and genetically encoded DNA origami has mimicked the functions of cell surface ligands, cell membrane, and nucleus for communication, protection, and gene expression, respectively. This rationally developed combination of folding and coating strategies for genetically encoded DNA origami presents a new avenue for the development of gene therapy.

Recent Advances in DNA Origami-Engineered Nanomaterials and Applications.

DNA nanotechnology is a unique field, where physics, chemistry, biology, mathematics, engineering, and materials science can elegantly converge. Since the original proposal of Nadrian Seeman, significant advances have been achieved in the past four decades. During this glory time, the DNA origami technique developed by Paul Rothemund further pushed the field forward with a vigorous momentum, fostering a plethora of concepts, models, methodologies, and applications that were not thought of before. This review focuses on the recent progress in DNA origami-engineered nanomaterials in the past five years, outlining the exciting achievements as well as the unexplored research avenues. We believe that the spirit and assets that Seeman left for scientists will continue to bring interdisciplinary innovations and useful applications to this field in the next decade.

DNA Nanomaterials-Based Platforms for Cancer Immunotherapy.

The past few decades have witnessed the evolving paradigm for cancer therapy from nonspecific cytotoxic agents to selective, mechanism-based therapeutics, especially immunotherapy. In particular, the integration of nanomaterials with immunotherapy is proven to improve the therapeutic outcome and minimize off-target toxicity in the treatment. As a novel nanomaterial, DNA-based self-assemblies featuring uniform geometries, feasible modifications, programmability, surface addressability, versatility, and intrinsic biocompatibility, are extensively exploited for innovative and effective cancer immunotherapy. In this review, the successful employment of DNA nanoplatforms for cancer immunotherapy, including the delivery of immunogenic cell death inducers, adjuvants and vaccines, immune checkpoint blockers as well as the application in immune cell engineering and adoptive cell therapy is summarized. The remaining challenges and future perspectives regarding the pharmacokinetics/pharmacodynamics, in vivo fate and immunogenicity of DNA materials, and the design of intelligent DNA nanomedicine for individualized cancer immunotherapy are also discussed.

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.

DNA-based enzymatic systems and their applications.

DNA strands with unique secondary structures can catalyze various chemical reactions and mimic natural enzymes with the assistance of cofactors, which have attracted much research attention. At the same time, the emerging DNA nanotechnology provides an efficient platform to organize functional components of the enzymatic systems and regulate their catalytic performances. In this review, we summarize the recent progress of DNA-based enzymatic systems. First, DNAzymes (Dzs) are introduced, and their versatile utilities are summarized. Then, G-quadruplex/hemin (G4/hemin) Dzs with unique oxidase/peroxidase-mimicking activities and representative examples where these Dzs served as biosensors are explicitly elaborated. Next, the DNA-based enzymatic cascade systems fabricated by the structural DNA nanotechnology are depicted. In addition, the applications of catalytic DNA nanostructures in biosensing and biomedicine are included. At last, the challenges and the perspectives of the DNA-based enzymatic systems for practical applications are also discussed.

A DNA-Based Plasmonic Nanodevice for Cascade Signal Amplification.

Here, we describe a DNA circuit-aided, origami nanodevice-based plasmonic system, which performs DNA-regulated, cascade amplification of faint chemical/biological signals. In this system, two gold-nanorods (GNRs) are co-assembled onto a DNA lock-containing, tweezer-like DNA origami template. Logic circuits serve as recognition and amplification elements for specific messengers, producing DNA keys for driving conformational changes of the plasmonic nanodevices. In the presence of input signals including nucleic acids, adenosines, chiral tyrosinamides or specific receptors expressed by tumor cells, the plasmonic nanodevices can be activated to perform dynamic structural motions, reporting robust responses via plasmonic circular dichroism (CD) spectral changes. This DNA nanodevice-based system provides a different design to enrich the strategies for constructing synthetic nanomachines, enabling the customized bottom-up nanostructure construction for sensitive biological signaling.

Stimuli-Responsive DNA Origami Nanodevices and Their Biological Applications.

DNA origami nanotechnology has provided predictable static nanoarchitectures and dynamic nanodevices with rationally designed geometries, precise spatial addressability, and marked biocompatibility. Multiple functional elements, such as peptides, aptamers, nanoparticles, fluorescence probes, and proteins, etc. can be easily integrated into DNA origami templates with nanoscale precision, leading to a variety of promising applications. Triggered by chemical/physical stimuli, dynamic DNA origami nanodevices can switch between defined conformations or translocate autonomously, providing powerful tools for intelligent biosensing and drug delivery. In this minireview, we summarize the recent progress of dynamic DNA origami nanodevices with desired reconfigurability and feasibility to perform multiple biological tasks. We introduce varieties of DNA nanodevices that can be controlled by different molecular triggers and external stimuli. Subsequently, we highlight the recent advances in employing DNA nanodevices as biosensors and drug delivery vehicles. At last, future possibilities and perspectives are also discussed.

Sparse deconvolution improves the resolution of live-cell super-resolution fluorescence microscopy.

A main determinant of the spatial resolution of live-cell super-resolution (SR) microscopes is the maximum photon flux that can be collected. To further increase the effective resolution for a given photon flux, we take advantage of a priori knowledge about the sparsity and continuity of biological structures to develop a deconvolution algorithm that increases the resolution of SR microscopes nearly twofold. Our method, sparse structured illumination microscopy (Sparse-SIM), achieves ~60-nm resolution at a frame rate of up to 564 Hz, allowing it to resolve intricate structures, including small vesicular fusion pores, ring-shaped nuclear pores formed by nucleoporins and relative movements of inner and outer mitochondrial membranes in live cells. Sparse deconvolution can also be used to increase the three-dimensional resolution of spinning-disc confocal-based SIM, even at low signal-to-noise ratios, which allows four-color, three-dimensional live-cell SR imaging at ~90-nm resolution. Overall, sparse deconvolution will be useful to increase the spatiotemporal resolution of live-cell fluorescence microscopy.

An RNA/DNA hybrid origami-based nanoplatform for efficient gene therapy.

Nucleic acid nanostructures are promising biomaterials for the delivery of homologous gene therapy drugs. Herein, we report a facile strategy for the construction of target mRNA (scaffold) and antisense (staple strands) co-assembled RNA/DNA hybrid "origami" for efficient gene therapy. In our design, the mRNA was folded into a chemically well-defined nanostructure through RNA-DNA hybridization with high yield. After the incorporation of an active cell-targeting aptamer, the tailored RNA/DNA hybrid origami demonstrated efficient cellular uptake and controllable release of antisenses in response to intracellular RNase H digestion. The biocompatible RNA/DNA origami (RDO) elicited a noticeable inhibition of cell proliferation based on the silencing of the tumor-associated gene polo-like kinase 1 (PLK1). This RDO-based nanoplatform provides a novel strategy for the further development of gene therapy.

DNA-based plasmonic nanostructures and their optical and biomedical applications.

In the past few decades, DNA nanotechnology has been developed a lot due to their appealing features such as structural programmability and easy functionalization. In the emerging field of DNA nanotechnology, DNA molecules are regarded not only as biological information carriers but also as building blocks in the assembly of various two-dimensional and three-dimensional nanostructures, serving as outstanding templates for the bottom-up fabrication of plasmonic nanostructures. By arranging nanoparticles with different components and morphologies on the predesigned DNA templates, various static and dynamic plasmonic nanostructures with tailored optical properties have been obtained. In this review, we summarized recent advances in the design and construction of static and dynamic DNA-based plasmonic nanostructures. In addition, we addressed their emerging applications in the fields of optics and biosensors. At the end of this review, the open questions and future directions of DNA-based plasmonic nanostructure are also discussed.

Strong Light-Matter Interactions in Chiral Plasmonic-Excitonic Systems Assembled on DNA Origami.

The exploitation of strong light-matter interactions in chiral plasmonic nanocavities may enable exceptional physical phenomena and lead to potential applications in nanophotonics, information communication, etc. Therefore, a deep understanding of strong light-matter interactions in chiral plasmonic-excitonic (plexcitonic) systems constructed by a chiral plasmonic nanocavity and molecular excitons is urgently needed. Herein, we systematically studied the strong light-matter interactions in gold nanorod-based chiral plexcitonic systems assembled on DNA origami. Rabi splitting and anticrossing behavior were observed in circular dichroism spectra, manifesting chiroptical characteristic hybridization. The bisignate line shape of the circular dichroism (CD) signal allows the accurate discrimination of hybrid modes. A large Rabi splitting of ∼205/∼199 meV for left-handed/right-handed plexcitonic nanosystems meets the criterion of strong coupling. Our work deepens the understanding of light-matter interactions in chiral plexcitonic nanosystems and will facilitate the development of chiral quantum optics and chiroptical devices.

A DNA origami-based aptamer nanoarray for potent and reversible anticoagulation in hemodialysis.

Effective and safe hemodialysis is essential for patients with acute kidney injury and chronic renal failures. However, the development of effective anticoagulant agents with safe antidotes for use during hemodialysis has proven challenging. Here, we describe DNA origami-based assemblies that enable the inhibition of thrombin activity and thrombus formation. Two different thrombin-binding aptamers decorated DNA origami initiates protein recognition and inhibition, exhibiting enhanced anticoagulation in human plasma, fresh whole blood and a murine model. In a dialyzer-containing extracorporeal circuit that mimicked clinical hemodialysis, the origami-based aptamer nanoarray effectively prevented thrombosis formation. Oligonucleotides containing sequences complementary to the thrombin-binding aptamers can efficiently neutralize the anticoagulant effects. The nanoarray is safe and immunologically inert in healthy mice, eliciting no detectable changes in liver and kidney functions or serum cytokine concentration. This DNA origami-based nanoagent represents a promising anticoagulant platform for the hemodialysis treatment of renal diseases.

A Tubular DNA Nanodevice as a siRNA/Chemo-Drug Co-delivery Vehicle for Combined Cancer Therapy.

Using the DNA origami technique, we constructed a DNA nanodevice functionalized with small interfering RNA (siRNA) within its inner cavity and the chemotherapeutic drug doxorubicin (DOX), intercalated in the DNA duplexes. The incorporation of disulfide bonds allows the triggered mechanical opening and release of siRNA in response to intracellular glutathione (GSH) in tumors to knockdown genes key to cancer progression. Combining RNA interference and chemotherapy, the nanodevice induced potent cytotoxicity and tumor growth inhibition, without observable systematic toxicity. Given its autonomous behavior, exceptional designability, potent antitumor activity and marked biocompatibility, this DNA nanodevice represents a promising strategy for precise drug design for cancer therapy.

A DNA nanodevice-based vaccine for cancer immunotherapy.

A major challenge in cancer vaccine therapy is the efficient delivery of antigens and adjuvants to stimulate a controlled yet robust tumour-specific T-cell response. Here, we describe a structurally well defined DNA nanodevice vaccine generated by precisely assembling two types of molecular adjuvants and an antigen peptide within the inner cavity of a tubular DNA nanostructure that can be activated in the subcellular environment to trigger T-cell activation and cancer cytotoxicity. The integration of low pH-responsive DNA 'locking strands' outside the nanostructures enables the opening of the vaccine in lysosomes in antigen-presenting cells, exposing adjuvants and antigens to activate a strong immune response. The DNA nanodevice vaccine elicited a potent antigen-specific T-cell response, with subsequent tumour regression in mouse cancer models. Nanodevice vaccination generated long-term T-cell responses that potently protected the mice against tumour rechallenge.

Cofactor-free oxidase-mimetic nanomaterials from self-assembled histidine-rich peptides.

Natural oxidases mainly rely on cofactors and well-arranged amino acid residues for catalysing electron-transfer reactions but suffer from non-recovery of their activity upon externally induced protein unfolding. However, it remains unknown whether residues at the active site can catalyse similar reactions in the absence of the cofactor. Here, we describe a series of self-assembling, histidine-rich peptides, as short as a dipeptide, with catalytic function similar to that of haem-dependent peroxidases. The histidine residues of the peptide chains form periodic arrays that are able to catalyse H2O2 reduction reactions efficiently through the formation of reactive ternary complex intermediates. The supramolecular catalyst exhibiting the highest activity could be switched between inactive and active states without loss of activity for ten cycles of heating/cooling or acidification/neutralization treatments, demonstrating the reversible assembly/disassembly of the active residues. These findings may aid the design of advanced biomimetic catalytic materials and provide a model for primitive cofactor-free enzymes.