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.

Efficient one-pot assembly of higher-order DNA nanostructures by chemically conjugated branched DNA.

Chemically conjugated branched DNA was successfully synthesized by a copper-free click reaction to construct sophisticated and higher-order polyhedral DNA nanostructures with pre-defined units in one pot, which can be used as an efficient nanoplatform to precisely organize multiple gold nanoparticles in predesigned patterns.

Light Responsive DNA Nanomaterials and Their Biomedical Applications.

DNA nanomaterials have been widely employed for various biomedical applications. With rapid development of chemical modification of nucleic acid, serials of stimuli-responsive elements are included in the multifunctional DNA nanomaterials. In this review, we summarize the recent advances in light responsive DNA nanomaterials based on photocleavage/photodecage, photoisomerization, and photocrosslinking for efficient bioimaging (including imaging of small molecule, microRNA, and protein) and drug delivery (including delivery of small molecule, nucleic acid, and gene editing system). We also discuss the remaining challenges and future perspectives of the light responsive DNA nanomaterials in biomedical applications.

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.

A DNA Origami-Based Gene Editing System for Efficient Gene Therapy in Vivo.

DNA nanostructures have played an important role in the development of novel drug delivery systems. Herein, we report a DNA origami-based CRISPR/Cas9 gene editing system for efficient gene therapy in vivo. In our design, a PAM-rich region precisely organized on the surface of DNA origami can easily recruit and load sgRNA/Cas9 complex by PAM-guided assembly and pre-designed DNA/RNA hybridization. After loading the sgRNA/Cas9 complex, the DNA origami can be further rolled up by the locking strands with a disulfide bond. With the incorporation of DNA aptamer and influenza hemagglutinin (HA) peptide, the cargo-loaded DNA origami can realize the targeted delivery and effective endosomal escape. After reduction by GSH, the opened DNA origami can release the sgRNA/Cas9 complex by RNase H cleavage to achieve a pronounced gene editing of a tumor-associated gene for gene therapy in vivo. This rationally developed DNA origami-based gene editing system presents a new avenue for the development of gene therapy.

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.

A DNA Origami-based Bactericide for Efficient Healing of Infected Wounds.

Bacteria infection is a significant obstacle in the clinical treatment of exposed wounds facing widespread pathogens. Herein, we report a DNA origami-based bactericide for efficient anti-infection therapy of infected wounds in vivo. In our design, abundant DNAzymes (G4/hemin) can be precisely organized on the DNA origami for controllable generation of reactive oxygen species (ROS) to break bacterial membranes. After the destruction of the membrane, broad-spectrum antibiotic levofloxacin (LEV, loaded in the DNA origami through interaction with DNA duplex) can be easily delivered into the bacteria for successful sterilization. With the incorporation of DNA aptamer targeting bacterial peptidoglycan, the DNA origami-based bactericide can achieve targeted and combined antibacterial therapy for efficiently promoting the healing of infected wounds. This tailored DNA origami-based nanoplatform provides a new strategy for the treatment of infectious diseases in vivo.

Ultrasensitive Detection of MicroRNA in Human Saliva via Rolling Circle Amplification Using a DNA-Decorated Graphene Oxide Sensor.

MicroRNAs (miRNAs) are a family of conserved small noncoding RNAs whose expression is associated with many diseases, including cancer. Salivary miRNAs are gaining popularity as noninvasive diagnostic biomarkers for cancer and other systemic disorders, but their use is limited by their low abundance and complicated detection procedure. Herein, we present a novel self-assembly approach based on rolling circle amplification (RCA) and graphene oxide (GO) for the ultrasensitive detection of miRNA21 and miRNA16 (miRNA oral cancer biomarkers in human saliva). First, target miRNA hybridizes with the RCA template. In the presence of DNA polymerase, the RCA reaction is induced and sequences matching the template are generated. Then, a nicking enzyme cuts the long ssDNA product into tiny pieces to obtain the amplified products. The DNA-decorated GO sensor was fabricated by preabsorbing the ssDNA fluorescence-labeled probe on the GO surface, resulting in fluorescence quenching. The DNA-decorated GO sensor could detect the amplified product via the self-assembly of dsDNA, leading to the desorption and recovery of the fluorescence-labeled probe. Under optimal conditions, the proposed system exhibited ultrasensitive detection; the detection limits of miRNA16 and miRNA21 were 8.81 and 3.85 fM, respectively. It showed a wide range of detection between 10 fM and 100 pM for miRNA16 and between 10 fM and 1 nM for miRNA16. It demonstrated high selectivity, distinguishing between 1- and 3-mismatch nucleotides in target miRNA. Overall, our proposed DNA-decorated GO sensor can accurately detect the salivary miRNAs and may potentially be used for the diagnosis and screening of early-stage oral cancer.

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.

Fluorescence-Amplified Origami Microneedle Device for Quantitatively Monitoring Blood Glucose.

Exploration of clinically acceptable blood glucose monitors has been engaging in the past decades, yet the ability to quantitatively detect blood glucose in a painless, accurate, and highly sensitive manner remains limited. Herein, a fluorescence-amplified origami microneedle (FAOM) device is described that integrates tubular DNA-origami nanostructures and glucose oxidase molecules into its inner network to quantitatively monitor blood glucose. The skin-attached FAOM device can collect glucose molecules in situ and transfer the input into a proton signal after the oxidase's catalysis. The proton-driven mechanical reconfiguration of DNA-origami tubes separates fluorescent molecules and their quenchers, eventually amplifying the glucose-correlated fluorescence signal. The function equation established on clinical examinees suggests that FAOM can report blood glucose in a highly sensitive and quantitative manner. In clinical blind tests, the FAOM achieves well-matched accuracy (98.70 ± 4.77%) compared with a commercial blood biochemical analyzer, fully meeting the requirements of accurate blood glucose monitoring. The FAOM device can be inserted into skin tissue in a trivially painful manner and with minimal leakage of DNA origami, substantially improving the tolerance and compliance of the blood glucose test.

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.

Stimuli-responsive nucleic acid nanostructures for efficient drug delivery.

Based on complementary base pairing, nucleic acid molecules have acted as engineerable building blocks to prepare versatile nanostructures with unique shapes and sizes. Benefiting from excellent programmability and biocompatibility, rationally designed nucleic acid nanostructures have been widely employed in biomedical applications. With the development of the chemical biology of nucleic acids, various stimuli-responsive nucleic acid nanostructures have been constructed by tailored chemical modification with multifunctional components. In this minireview, we summarize the representative and latest research about the employment of stimuli-responsive nucleic acid nanostructures for drug delivery in response to endogenous and exogenous stimuli (redox gradient, pH, nuclease, biomacromolecule, and light). We also discuss the broad prospects and remaining challenges of nucleic acid nanotechnology in biomedical applications.

A covalently conjugated branched DNA aptamer cluster-based nanoplatform for efficiently targeted drug delivery.

Targeted delivery of therapeutic drugs is essential for precise treatment of various diseases to reduce possible serious side-effects. A screened DNA aptamer has been widely developed for active targeting delivery. Herein, we report a facile strategy for the construction of a branched DNA aptamer cluster-based nanoplatform for efficiently targeted drug delivery. In our design, the terminal-modified DNA aptamer can be covalently conjugated to form a branched aptamer cluster by click reaction easily. The branched aptamer cluster-modified DNA tetrahedron (TET) demonstrates highly targeted cellular uptake with the modification of only one site. After loading the chemotherapeutic drug (doxorubicin, DOX), the DNA aptamer cluster-based nanoplatform elicits a remarkable and selective inhibition of tumor cell proliferation by much-enhanced targeted delivery. This covalently conjugated branched DNA aptamer cluster-based nanoplatform provides a new strategy for the development of targeted drug delivery.

Chemically modified DNA nanostructures for drug delivery.

Based on predictable, complementary base pairing, DNA can be artificially pre-designed into versatile DNA nanostructures of well-defined shapes and sizes. With excellent addressability and biocompatibility, DNA nanostructures have been widely employed in biomedical research, such as bio-sensing, bio-imaging, and drug delivery. With the development of the chemical biology of nucleic acid, chemically modified nucleic acids are also gradually developed to construct multifunctional DNA nanostructures. In this review, we summarize the recent progress in the construction and functionalization of chemically modified DNA nanostructures. Their applications in the delivery of chemotherapeutic drugs and nucleic acid drugs are highlighted. Furthermore, the remaining challenges and future prospects in drug delivery by chemically modified DNA nanostructures are 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.

DNA Nanodevices: From Mechanical Motions to Biomedical Applications.

Inspired by molecular machines in nature, artificial nanodevices have been designed to realize various biomedical functions. Self-assembled deoxyribonucleic acid (DNA) nanostructures that feature designed geometries, excellent spatial accuracy, nanoscale addressability, and marked biocompatibility provide an attractive candidate for constructing dynamic nanodevices with biomarker- targeting and stimuli-responsiveness for biomedical applications. Here, a summary of typical construction strategies of DNA nanodevices and their operating mechanisms are presented. We also introduced recent advances in employing DNA nanodevices as platforms for biosensing and intelligent drug delivery. Finally, the broad prospects and main challenges of the DNA nanodevices in biomedical applications are also discussed.