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.


Introduction
The precise control of molecular self-assemblies at the nanoscale and microscale is a consistent pursuit of nanoscience and technology.In the field of structural DNA nanotechnology, DNA molecules, in addition to their classic, biological role as carriers of genetic information, are regarded as building blocks for creating exquisite architectures with well-defined sizes and shapes, based on predictable base-pairing rules. [1]Various approaches, including tile-based assembly, [2] enzyme-derived rolling circle amplification/transcription, [3] and DNA origami techniques, [4] have been developed for fabricating DNA nanostructures.These research advances in the design and assembling of artificial DOI: 10.1002/adma.202301035DNA structures and devices have laid the foundation for various applications, [1,4b] especially in the biological and biomedical space. [5]mong the various assembling methodologies, DNA origami is a promising branch of DNA nanotechnology for constructing biodegradable and biocompatible materials with predesigned geometries, precise addressability, and versatile programmability.A DNA origami nanostructure, usually ranging from tens of nanometers to sub-micrometers, can be folded by a scaffold strand (typically bacteriophage genomic DNA, ≈7000 nucleotides long) and hundreds of staple strands (≈20-60 nucleotides long).During the self-assembly process, the staples hybridize to specific sites of the scaffold DNA strand, folding and knitting it into a pre-designed structure.Through an annealing process, the resulting DNA origami structures with programmed and uniform geometries can be synthesized in high yields.For example, DNA nanocontainers with cavities can be designed and fabricated with high precision, generating an internal space that can hold molecular payloads free from interference from the external environment.More importantly, these homogenous DNA origami nanostructures present entirely addressable surfaces that can be utilized as nanoscale drawing boards, where multiple moieties with desired functions (small molecule dyes or drugs, peptides, proteins, nucleic acids, organic/inorganic nanoparticles, and other targeting ligands or therapeutic components) can be precisely positioned with rationally designed numbers and patterns.The predesigned structural features of the DNA origami, including sizes, shapes, surface modifications, may also affect and regulate the process of in vivo biological barrier penetration.Stimuli-responsive dynamic DNA origami devices capable of undergoing fine-tuned structural reconfiguration can also be constructed to enable the release of loaded cargos under specific stimuli at desired sites.
Clinical developments in disease treatment have benefited from the rapid development of drug delivery strategies. [6]To date, scientists have developed various drug delivery systems both to shield cytotoxic small molecules (e.g., doxorubicin, paclitaxel) from nonspecific tissue distribution and to protect highly unstable macromolecule-based therapeutics (e.g., mRNA, siRNA) from degradation.These strategies can result in the maximal delivery of drugs to the appropriate biological targets while minimizing undesirable effects in healthy tissues.Taking advantage of their prominent structural properties, various DNA origami is folded into an arbitrary origami shape with the aid of multiple, short staple DNA strands.As a candidate for innovative nanocarrier, DNA origami holds distinct advantages, e.g., inherent biodegradability and biocompatibility, arbitrary and high-yield geometries, tailored functionality, and fine-tuned structural reconfiguration.Versatile functional DNA origami nanostructures loaded with active pharmaceutical ingredients (APIs) have been designed and constructed.With their advantageous structural properties, DNA origami technology has yielded various promising platforms for optimized cargo delivery and disease therapy.
nanoarchitectures have been developed for drug delivery, with the goals of protecting therapeutic agents from rapid clearance and degradation, helping drugs to cross multiple biological barriers, specifically targeting malignant tissues/cells, and controlling drug release profiles (Figure 1).In this Perspective, we will highlight the recent advances in the application of DNA origami as an innovative class of drug delivery systems to modulate the behaviors of bioactive agents and enable safe and effective disease treatment.

Static DNA Origami Structures
Self-assembly technology has provided scientists with flexible and robust approaches to manipulate molecules at the nanoscale and microscale.Static 2D or 3D DNA origami nanostructures have been used as drawing boards to organize multiple components (including enzymes, antigens, peptides, nucleic acids, amphiphilic molecules, etc.) with nanoscale precision. [7]Based on these predesigned and customized nanostructures, exquisite assemblies can perform fine-tuned biological functions, such as regulation of enzyme activities, stimuli-responsive protein coverage for triggered antigen binding, [8] guiding, and manipulation of biological membranes. [9]an's group presented a series of enzyme-DNA origami assemblies, [10] which consisted of spatially addressable origami templates and precisely organized enzyme molecules (e.g., glucose oxidase [GOx], horseradish peroxidase [HRP], glucose6phosphate dehydrogenase [G6pDH], and lactate dehydrogenase [LDH]).10b] Sequential enzymatic cascade and substrate channeling were further investigated based on DNA origami techniques by Klein et al. [11] For real-time imaging of the enzyme cascade, an artificial origami raft on a supported lipid bilayer (SLB) was reported by Sun et al. [12] Next, Xu et al. advanced this real-time single-molecule characterization method, describing the correlation between catalytic performance and docking sites. [13]Recently, Rosier et al. developed DNA-origami-based caspase-9 assemblies that function as artificial apoptosomes (Figure 2A). [14]The authors found that the catalytic capacity of the synthetic protein assemblies was induced by proximity-driven dimerization of caspase-9, and could be further improved by clustering three and four caspase-9 monomers.
DNA-origami-organized antigen multimers have also been designed for the regulation of immune response.Veneziano et al. presented DNA frameworks and nanorods to sitespecifically arrange antigen molecules (HIV-1 envelope glycoprotein antigen gp120, eOD-GT8) for manipulating B-cell activation (Figure 2B). [15]The parameters of the origami-antigen assemblies (e.g., the antigen copy number, the inter-antigen separation, and rigidity of DNA origami templates, etc.) were found to be significant for B-cell receptor activation.They demonstrated that five eOD-GT8 molecules displayed on DNA icosahedron were able to induce B-cell response.The cellular response could not be further enhanced by increasing the valency of antigen copies.
More importantly, an intermolecular spacing of 25-30 nm for antigen copies and relatively rigid origami templates could elicit robust B-cell triggering.Sun et al. constructed 2D triangular DNA origami nanostructures displaying peptide-MHC (pMHC) multimers to affect T-cell regulation (Figure 2C). [16]The pMHC stoichiometries and inter-ligand spacing were essential for the assemblies' biological functions.Both increasing the number of pMHC moieties and decreasing the spacing between two ligands improved the interaction of pMHC multimers with T-cell receptors (TCRs).With the use of these origami-based pMHC multimers in mice, the authors demonstrated the precise analysis of antigen-specific T cells expressing low-affinity TCRs that were difficult to detect using an equivalent number of free tetramers.Recently, the same group also described DNA origami nanostructures co-loaded with pMHC and an anti-CD28 antibody (aCD28) as artificial antigen-presenting cells (aAPCs) for adoptive T cell therapy.The aCD28 molecules, as co-stimulatory ligands, were located at the vertices of the triangular DNA origami scaffold, while pMHC, as TCR ligands, were assembled at the three edges of the DNA template, forming the origami-based aAPC.The authors studied the effects of ligand organization by origami scaffolds on T-cell stimulation.They concluded that shorter inter-pMHC spacing can induce a more robust T-cell activation.At the animal level, the origami-based aAPCs triggered antigen-specific and effective tumor growth inhibition. [17]sing DNA strands as ligands for the modulation of biological function, Comberlato et al. demonstrated an approach to construct 2D, disc-like DNA origami precisely decorated with unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs), a type of ligand for Toll-like receptor 9 (TLR 9). [18]Similar to the abovementioned static origami systems, the pre-designed ligand patterns of the immunostimulatory CpG sequences affected immune stimulation on the murine macrophage cells, Raw 264.7.When CpGs were pinned with an inter-ligand spacing of 7 nm, which suitably matches the dimer structure of TLR9, the potent activation of immune signaling was observed.Zhao et al. introduced another origami strategy for guiding functional nucleic acids for effective and safe anticoagulation in vivo by fabricating thrombin-binding aptamer nanoarrays based on DNA origami rectangular nanostructures (Figure 2D). [19]On the addressable surface of a DNA origami sheet, two types of aptamers for binding thrombin at different exosites were arranged with precisely controlled interaptamer distances.When the inter-molecule spacing was set to 5.4 nm, approximately matching the dimension of the thrombin molecules (≈4 nm), a bivalent aptamer array with prominent protein binding affinity was achieved.Upon incubation with human plasma/whole blood or administration through intravenous injection in mice, the origami-based nanoarrays induced effective anticoagulation, which was also reversible and could be neutralized by aptamer-complementary strands.
Besides building nanoplatforms displaying with oligonucleotides, long and flexible genes can also be feasibly and efficiently integrated into DNA origami.Liu et al. developed a kitelike DNA origami structure to deliver p53 genes as therapeutic cargos into tumor cells. [20]Linear, sticky-end-containing tumor suppressor gene sequences were prepared using extended forward primers and polymerase chain reaction (PCR) technology.After the large gene portions loaded site-selectively, the origami structures acted as non-viral nanovectors that facilitated the gene delivery, intracellular controlled release as well as enhanced gene expression for inhibiting tumor growth.Without using gene fragments as positioning guest molecules, Kretzmann et al. created gene-folded DNA origami nanostructures and transported them into mammalian cells for gene expression. [21]As a target gene for intracellular delivery, an enhanced green fluorescent protein (EGFP)-encoded phagemid was constructed and used for scaffold ssDNA production.Subsequently, the designed scaffold DNA strands were used to fabricate corresponding DNA origami structures.Using electroporation technology, the gene-folded DNA origami objects were delivered into the human embryonic kidney 293T (HEK293T) cells and demonstrated enhanced EGFP expression.Another successful design and application for genetically encoded DNA origami was provided by Wu et al. [22] Using p53 gene as a PCR template, the complementary sense and antisense DNA strands were prepared as two parts of the origami scaffold.After magnetic bead separation and subsequent folding process with the corresponding helper strands, the two parts of DNA origami were achieved and then hybridized to form the final genetically encoded origami object.Precise lipid decoration was used to enhance the cellular delivery of the gene-folded origami structures and their gene expression.In tumor-bearing mice, the lipid-encapsulated DNA origami elicited a potent upregulation of p53 expression and efficient tumor growth inhibition (Figure 2E).
Masubuchi et al. reported a logic chip to control gene expression. [23]The authors constructed a 2D DNA origami sheet to precisely position SNAP f protein-fused T7 RNA polymerase (RNAP) molecules with their substrates (endogenous genes).In their report, several parameters, such as intermolecular distances between the enzymes and target genes, the tethering direction of the genes, and the length and rigidity of the linkers, all impacted the transcriptional activity of the nanochips.Hahn et al. constructed a 3D origami barrel containing enzymes/substrates for RNA production and processing. [24]The multiple moieties for their corresponding functions, such as RNA-producing components (consisting of DNA templates and RNA polymerases) and RNA-processing units (such as RNA endonucleases), were precisely positioned in the inner cavity of the DNA barrel, thus ) pMHC multimers on triangular DNA origami for T cell regulation and detection.D) Thrombinbinding aptamer nanoarrays on DNA origami for controllable coagulation.E) A genetically encoded DNA origami for gene therapy.F) DNA-origamienabled liposomes for the study of protein-mediated lipid transfer.A) Adapted with permission. [14]Copyright 2020, The Authors, published by Springer Nature.B) Adapted with permission. [15]Copyright 2020, The Authors, published by Springer Nature.C) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0). [16]Copyright 2022, The Authors, published by Springer Nature.D) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0). [19]Copyright 2022, The Authors, published by Springer Nature.E) Adapted with permission. [22]Copyright 2023, American Chemical Society.F) Adapted with permission. [31]opyright 2019, The Authors, published by Springer Nature.avoiding environmental interference.Within this origami-based nanofactory, multiple copies of premature RNA transcripts were generated and subsequently cleaved by the integrated RNA endonucleases to form mature RNA molecules.
Lipid membranes and vesicles are important components of living organisms and play essential functions in biological processes.Manipulating and guiding amphiphilic molecules to form artificial membranes and vesicles can provide us with significant information for understanding life.In 2014, inspired by the natural cytoskeletal-membrane protein-lipid bilayer structures, Dong et al. developed a strategy to use ssDNA-modified gold nanoparticles as assembly frameworks to guild synthetic amphiphilic molecules to form vesicle structures with programmed geometries and dimensions. [25]Monodispersed and shape-defined membranes and vesicles were further constructed using 2D or 3D DNA origami nanostructures as frames. [26]Yang et al. presented an approach to construct artificial liposomal vesicles of controlled sizes and shapes with nanometer precision using DNA origami techniques. [27]Through hybridization with complementary ssDNA on the interior surface of the origami templates, DNA ring structures were fabricated and decorated with multiple lipidated ssDNA strands.Subsequently, the extra lipids and detergents were mixed with lipid molecules-containing origami rings, producing liposomal vesicles with determined sizes by the well-folded origami templates.Based on this strategy, they further developed reconfigurable DNA nanocages to guide the formation of liposomes with designer geometries. [28]y transcribing the structural information of DNA cage templates to liposomes with high fidelity, differently shaped liposomal structures were successfully generated, including widthdefined straight tubes, helical tubes, tubule arrays, etc.In 2018, the same group constructed helical, rod-like DNA origami motifs, which were polymerized by linker strands into left-handed DNA nanosprings.These large structures were then used as lipid-guided templates for vesicle tubularization by attaching amphiphilic peptides as membrane anchors along the inner surface of the origami. [29]They also studied the impact of structural parameters, such as the thickness and rigidity of DNA templates and the density of amphipathic anchoring molecules on vesicle tubulation. [30]The results showed that reducing origami rigidity and increasing the anchor density would be effective in generating structurally well-defined lipid vesicles.In 2019, Bian et al. used the origami platform to precisely guide liposome formation and studied protein-mediated lipid transfer between bilayers (Figure 2F). [31]In this work, they followed the abovementioned approaches to construct the monomeric, DNA-ring-templated liposomes and utilized six helix-bundle DNA rod scaffolds with sticky ends to form the dumbbell-like dimers.The distances of DNA ring-tethered liposomes can be precisely controlled by the lengths of the DNA rods.Subsequently, the origami-templated, liposome-containing dimers were used to investigate the impact of bilayer distances on lipid transport by the synaptotagminlike mitochondrial lipid-binding protein (SMP) domain of extended synaptotagmin 1 (E-Syt1).Besides demonstrating that SMP-mediated lipid transfer can occur over distances beyond its length, this work also provided a simple way to study biological processes between plasma membranes.
These static, origami-based assemblies provide innovative platforms to study the essential functions of multi-enzymes/antigens/oligonucleotides/genes/lipid complexes involved in biological processes, including inflammation, immunity, cell death, and molecular transportation.In the future, origami-based nanochips or nanofactories may further be optimized for producing therapeutic macromolecules.

Dynamic DNA Origami Structures
With the development of DNA nanotechnology, the design of dynamic DNA-origami-based devices and machines has seen significant advancements.Dynamic DNA nanostructures, including nanoboxes with openable lids, [32] 3D devices and robots, [33] "domino" nanoarrays, [34] robotic origami arms, [35] etc., have been reported to facilitate desired structural reconfiguration.The unique properties of the dynamic DNA origami afford nanoassemblies the ability to respond to multiple internal/external stimuli, which is essential for programmed functions.
As innovative tools with custom 3D geometries, dynamic DNA origami structures have been designed for high-resolution imaging to track key biological events (Figure 3A). [36]Kosuri et al. constructed a fluorophore-modified, cross-shaped DNA rotor, consisting of four blades, to monitor enzyme-induced DNA rotation at the single-molecule level with a time resolution of milliseconds.The motor protein was connected to the central region of the DNA origami rotor through a double-stranded DNA (dsDNA) segment that functioned as a substrate for the DNA-interacting enzyme.Using the origami rotor-based imaging approach, DNA rotation resulting from unwinding by helicase-involved DNA repair was measured and tracked.A series of significant events during DNA rotations (e.g., RecBCD initiation, pausing and backtracking during DNA unwinding, and the rotational steps during transcription by RNAP) were detectable, thus exemplifying a robust, DNA-origami-based tool for studying and monitoring dynamic processes, at a high resolution, in biological systems.
A variety of controlling strategies can be incorporated into DNA origami nanostructures, realizing dynamic performance under specific internal/external signals.This type of dynamic DNA origami structure has been utilized for the detection of specific chemical/biological messengers.For instance, Liu et al. constructed gold nanorod (GNR)-decorated, tweezer-like DNA origami nanodevices as chiral probes, in which DNA-based controlling components (containing disulfide bonds or specific aptamer sequences responsive to adenosine) were integrated. [37]pon the addition of the exterior signal molecules, glutathione (GSH) or adenosine, the controlling components became "unlocked," allowing the separation of the two arms of the DNA origami tweezers.The structural reconfiguration of the nanodevices was converted to a plasmonic circular dichroism (CD) signal output, realizing the recognition and detection of the corresponding molecular inputs.Recently, the same group advanced their origami-based plasmonic nanodevices by including a DNA logic circuit to recognize and amplify weak biological signals (Figure 3B). [38]Using such origami-based, signal-amplifying platforms, trace amounts of chemical/biological messengers, including small molecules or living cancer cells that express particular receptors, can be identified and detected, presenting a feasible approach for studying sensitive biological signaling.
Dynamic DNA origami structures can also behave as switchable containers for enzymes to precisely regulate enzymesubstrate interactions.Openable origami containers were utilized by Grossi et al., who located enzyme molecules in the origami cavities. [39]After the addition of corresponding DNA strands to close the edges of the framework, the DNA containers became sealed, shielding the enzymes from the physiological environment.In the presence of key strands, the closed origami containers could be switched to the open state, allowing the enzymes to access their substrates for catalysis.In addition to DNA origami containers that respond to corresponding DNA strands for structural unlocking, pH-manipulated DNA origami nanocapsules for studying enzyme-catalyzed reactions were reported by Ijas et al. (Figure 3C). [40]DNA locking strands containing pH-responsive sequences were incorporated into origami structures, enabling pH-controlled, reversible opening/closing reconfiguration of the nanocontainers to precisely regulate catalysis.The impact of the DNA containers on the enzyme-origami systems was recently studied by Saccà et al. [41] The authors demonstrated that the enzymatic reaction rates were affected by DNA/enzyme binding affinity and DNA/substrate electrostatic interactions.More importantly, DNA frameworks were proven to be not only non-functional scaffolds but also active participants in the reactions, providing alternative kinetic routes for substrate hydrolysis.
Besides serving as containers for biological macromolecules, DNA origami nanostructures have been designed as traps for viral particle sequestration and viral infection inhibition. [42]Based on this structural design, Engelen et al. provided switchable, multilayered, icosahedral DNA origami shells, which were formed via shape-complementary stacking of 20 identical triangular origami units and stabilized with IgG "molecular staples".After adding soluble antigens, the IgG staples were displaced from the icosahedral origami, triggering structural disassembly (Figure 3D). [43]his work provided a unique strategy by using DNA origami nanodevices as novel carriers for the on-demand release of molecular cargo.
DNA-origami-based dynamic architectures have been used to engineer nanoscale gates that can act as artificial channels to control molecular exchange.2012 saw the first development of twolayered origami DNA nanostructures as synthetic transmembrane channels by Langecker et al. [44] A stem-like DNA structure with a 2 nm cavity was designed to penetrate a lipid membrane for the passage of ions or single DNA molecules.On the outside of the stem module, a barrel-shaped DNA cap with cholesterol components was utilized to anchor the lipid bilayer.Based on this pioneered work, significant progress has been made in constructing large-sized, active DNA nanopores and nanoholes with sens-ing properties.Thomsen et al. created a DNA nanopore with an inner diameter of 9.6 nm for controlling the flow of molecules across lipid bilayers.Three programmable and dynamic DNA flaps, locked by DNA hybrids, were designed, which can be used to shield the lipidated decorations from the aqueous environment to avoid hydrophobicity-driven aggregation.The flap modules were triggered to open when opening signals based on strand displacement mechanism were present, resulting in the insertion of the DNA nanopore into the lipid membrane.A size-selective gating system for the translocation of macromolecules through the artificial channels was achieved by introducing 20 kDa PEG polymers as molecular plugs to the internal nanopores. [45]In another dynamic DNA origami nanohole system, DNAzymes or light were introduced as active triggers to program the unlocking structural changes that produced confined nanocavities. [46] similar and advanced reversible gating system based on DNA origami plate was developed by Dey et al. in 2022.[47] A 70 nm × 70 nm DNA origami plate containing a large-sized pore of 20.4 nm × 20.4 nm was constructed.An integrated DNA lid covered the pore, which was designed to be reversible and tunable based on a strand displacement mechanism for closing and opening.Multiple hydrophobic cholesterol moieties around the pore on the bottom surface of the plate were used to anchor the entire DNA nanopore structure for insertion into the lipid bilayer membrane.With the aid of the much wider pore and the sensitive lid, precisely timed, stimulus-controlled transport of molecular cargos, including small-molecule dyes and folded proteins, across lipid membranes was achieved (Figure 3E).
Another class of dynamic DNA origami nanostructures and nanodevices have been constructed and used to study minute forces and mechanical properties of biomolecules.Nickels et al. developed a DNA-origami-based force clamp, [48] which consists of a single-stranded DNA (ssDNA) that served as an entropic spring connecting to two immobile anchor points of a bracketshaped DNA origami structure.The length of ssDNA spring could be precisely adjusted, which affected the entropic force acting on the nanoscopic system.As a force clamp, the ssDNA spring portion could exert minor tensions in the low piconewton range on a molecular system, e.g., TATA-binding protein (TBP), which is important in transcriptional regulation and chromosome organization.Subsequently, single-molecule Förster resonance energy transfer (FERT) was utilized to detect the corresponding conformational transitions of the molecular system.A similar origami force clamp was created by Kramm et al. to study TBP-induced DNA bending. [49]In this advanced clamp, a double-stranded promoter region containing TATA-box element was included in the entropic spring, and the fluorescent dye pair was modified for FRET sensing.Once the TBP Figure 3. Dynamic DNA origami devices and containers.A) DNA origami rotors for high-resolution imaging and tracking biological events at the singlemolecule level.B) DNA logic circuit-aided, tweezer-like plasmonic DNA nanodevices for amplifying weak biological signals.C) pH-manipulated DNA origami nanocapsules for studying enzyme reactions.D) Antigen-triggered, switchable multilayer icosahedral DNA origami shells.E) DNA-origamibased large and gated channel.F) DNA origami force clamp for monitoring TBP-induced DNA bending.A) Adapted with permission. [36]Copyright 2019, The Authors, published by Springer Nature.B) Adapted with permission. [38]Copyright 2022, Wiley-VCH.C) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0). [40]Copyright 2019, The Authors, published by American Chemical Society.D) Adapted with permission. [43]Copyright 2021, The Authors, published by American Chemical Society.E) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0). [47]Copyright 2022, The Authors, published by Springer Nature.F) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0). [49]opyright 2020.recognized promoter dsDNA, the FERT-pair-containing DNA spring of the origami clamp bent, thereby reducing the distance between the fluorophores leading to an increase in FRET efficiency.The nanoscopic force clamp was used to monitor the assembly of human initiation complexes in the RNA polymerase (RNAP) II and RNAP III systems at the single-molecule level under piconewton forces (Figure 3F).Funke et al. created a tweezerlike dynamic DNA origami nanostructure as a spectrometer to study the forces between two nucleosomes. [50]Two nucleosomes were attached site-specifically onto the inner sides of the two arms of the DNA origami tweezers.Direct single-particle imaging with transmission electron microscopy (TEM) and FRET monitoring were utilized to study the nucleosome-nucleosome distances, enabling a distance-dependent energy landscape for nucleosome pair interactions.These advances suggest that DNA origami devices can be designed as powerful tools to study the molecular interactions between complex protein assemblies, providing direct and significant information on biological processes.
These dynamic origami-based devices, containers, nanopores, force clamps, and spectrometers have been engineered and utilized for tracking biological events, sensing chemical/biological messengers, manipulating enzymatic reactions, controlling molecular changes, and exploring mechanical properties of macromolecules.In molecular gating systems or force-sensing spectrometers, DNA origami work as active participants in biological or biomimetic processes.It is likely that more sophisticated, intelligent nanodevices will be adapted to generate a wide range of biological and biomedical applications, such as biosensing and drug delivery, in the future.

Intelligent DNA-Origami-Based Platforms for Drug Delivery
To date, DNA origami has proven to be a powerful and versatile platform for precise localizing multiple desired functional components (e.g., targeting ligands, therapeutic payloads, and stimuli-responsive motifs) in rationally designed numbers and patterns, giving rise to a variety of programmable and intelligent drug delivery applications.In particular, dynamic DNA origami structures offer many advantages over static designs.Sophisticated 3D vessel-like structures can be designed as drug vehicles with payload docking sites on the inner sides or in the cavities, protecting the assembled molecular cargos from being interfered with the complicated biological environment.Specific targeting/recognizing molecules, such as aptamers, peptides, antibodies, etc., can be feasibly incorporated into the outer surface of the nanocontainers, which would enhance the in vivo delivery and efficiently increase the amount of therapeutic payloads in the target regions.The dynamic design of the drug vehicles with internal/external stimuli-triggered reconfiguring properties can be customized, enabling a controlled drug release to elicit the desired functions at the desired sites.
In 2012, the pioneering work of a smart DNA origami robot as drug vehicle was reported by Douglas et al.The authors designed a dynamic DNA nanobarrel to carry multiple molecular payloads within its inner cavity and facilitate the payloads' delivery to target cell membranes (Figure 4A). [51]Aptamer-containing, logicgated DNA "locks" were designed to fold the DNA barrels into a closed state, and specifically respond to receptors on the target cell surface, thus exposing the loaded cargos to manipulate cell signaling.Based on this report of DNA robots working on cell surfaces, Amir et al. advanced the application of DNA nanorobots in living insects. [52]Using Blaberus discoidalis as an animal model, the group applied DNA origami robots to perform complicated tasks, including various logic-gated operations (AND, OR, XOR, NAND, NOT, CNOT, and a half adder), within the biological environment.Subsequently, the same group showed that DNA nanorobots in living insects can be temporally manipulated by recorded human electroencephalogram (EEG) patterns associated with cognitive states. [53]n 2018, Li et al. presented thrombin-loaded DNA nanorobots and utilized them in mice to precisely occlude tumor vessels to deprive tumors of nutrients (Figure 4B). [54]Thrombin molecules, as unique therapeutics, were tagged with ssDNA handles and localized to 2D DNA origami rectangles.AS1411 aptamer-containing DNA locking pairs were then used to seal the edges of the DNA sheets, forming tubular structures with the thrombin molecules inside the cavities.When the protein-loaded DNA nanotubes encountered nucleolins specifically expressed on tumor vessels, the intelligent nanocarriers were activated and opened to reveal the thrombin molecules, which triggered a coagulation cascade in situ.Intravenous delivery of the DNA robots to tumor-bearing mice induced thrombosis precisely within tumor vessels, eliciting potent tumor necrosis and regression.In safety assessments, the nanorobot-based selective occlusion therapeutic did not produce observable toxicity or immunogenicity in vivo, demonstrating its potential as a safe and effective cancer therapy.
In 2021, Wang et al. reported a stimuli-responsive DNA nanodevice for the co-delivery of chemo-drugs and RNA therapeutics in vivo. [55]For the two types of therapeutic cargos, doxorubicin molecules were intercalated into the DNA helix of the nanodevice, while small interfering RNA (siRNA) sequences that downregulated Bcl-2 and P-glycoprotein were loaded into the interior space of the 3D nanodevice through hybridization.Disulfide bond-containing DNA strands were used to lock the edges of the DNA nanodevice.Once internalized by tumor cells, the attached DNA locks of the nanodevices could be cleaved by GSH, converting the device structures to the open state and releasing the siR-NAs.The DNA nanodevice-based chemo/siRNA combined therapy elicited effective RNA interference and tumor growth inhibition without observable systematic toxicity.
Another novel DNA nanocarrier designed to deliver immunostimulant components as a cancer vaccine was developed by Liu et al. (Figure 4C). [56]DNA sequences with low-pH responsive function served as locks to form reconfigurable DNA nanotubes, while antigen peptides and nucleic acid adjuvants (CpG sequences and dsRNA) were loaded inside the inner spaces of the tubular structures.After subcutaneous administration in vivo, the tubular nanodevices accumulated in dendritic cells (DCs) of draining lymph nodes (dLNs).Specifically responding to the mildly acidic environment in the lysosomes of DCs, the nanodevices underwent reconfiguration and exposed their molecular payloads to trigger an immune response.In melanoma and colon carcinoma mouse models, antigen-specific T-cell responses were elicited by vaccination with the DNA nanodevice, and potent tumor regression and long-term T-cell memory were observed.All these origami-based intelligent systems are inspirations for the design of new delivery nanoplatforms for multiple therapeutic  [51] Copyright 2012, The American Association for the Advancement of Science.B) Adapted with permission. [54]Copyright 2018, Springer Nature.C) Adapted with permission. [56]Copyright 2020, The Authors, published by Springer Nature [ components to realize safe, efficient, and personalized disease treatment.

Conclusions and Future Perspectives
Since the first description of DNA origami in 2006, [4a] structural DNA nanotechnology has been utilized to design and create architectures with a variety of well-defined, nanoscale/microscale homogenous structures.These research advances have led to the transition of novel DNA-origami-based assemblies from academic interests to innovative nanomaterials with programmable functionalities for various applications, especially in biological and biomedical fields.
In contrast with the classic nanoscale delivery systems, including lipid-based nanoparticles, polymeric nanoparticles, and metallic nanoparticles, self-assembled DNA-origami-based drug carriers provide engineered nanoplatforms that can load and release drugs in a precisely controlled manner. [57]Small molecular drugs, such as nucleoside analogs (floxuridine, gemcitabine, decitabine, etc.), can be covalently and quantitively incorporated into the nucleic-acid-based structures by replacing nucleoside units. [58]DNA origami nanocarriers with rationally controlled drug release kinetics have been achieved by introducing nanostructures with different degrees of global twist or by precisely controlling drug loading conditions (pH, concentration of Mg 2+ , and drug molecules). [59]In addition, functional nucleic acids, including antisense sequences (ASOs), small interference RNAs (siRNAs), micro RNAs (miRNAs), etc., can be integrated into nanovehicles by direct extension or hybridization with complementary capture strands at the positions of the fully addressable DNA structures.For macromolecular drugs, including peptides and proteins, ssDNA molecules can be conveniently attached to their surfaces, allowing the conjugates to be presented to DNA origami nanoplatforms via site-specific hybridization.Several strategies, including nucleic acid strand displacement, [60] enzymatic or chemical cleavage of covalent bonds [55] provide efficient and controllable ways for releasing such macromolecular payloads.
Though significant progress has been achieved in constructing numerous DNA assemblies with programmable functions, several crucial issues must be addressed to further advance DNA-based nanomaterials toward sophisticated biomedical applications and future clinical practice (Figure 5).For instance, although constructed by biocompatible DNA molecules, the biosafety and potential immunogenicity of DNA nanoassemblies should be key considerations.Since the DNA starting materials, long, single-stranded scaffold DNA molecules, are usually derived from bacteriophage-infected Escherichia coli via fermentation in bioreactors, [61] any residual lipopolysaccharide endotoxin in the resulting DNA origami structures may trigger immunostimulatory effects in mammalian organisms.Recent studies have shown that endotoxin-removal procedures can and should be incorporated into the process of scaffold DNA preparation and purification. [62]After mass production and endotoxin removal, non-drug loaded DNA origami structures elicited a modest proinflammatory immune response that diminished over time and were nontoxic in mice after systematic administration at a relatively high dose (12 mg kg −1 , ≈500 nm origami, five total injections), indicating that DNA origami represents a promising and safe platform for drug delivery.Although pure DNA origami demonstrated limited immune activation effects, multiple stimulatory nucleic acids that trigger innate immune responses through the activation of toll-like receptors (TLRs), such as CpG DNA motifs, single-stranded RNA (ssRNA), and double-stranded RNA (dsRNA), can be quantitatively attached to the DNA nanoarchitectures for promoting robust and designed immune activation.After fine-tuning their immunogenicity, innovative adjuvants for both immunotherapy [56] and the study of immune regulatory mechanisms [18] have been achieved.Furthermore, by organizing and manipulating antigen/adjuvant components with nanoscale distance control on the addressable origami surfaces, a series of origami-based architectures for immune cell recognition and activation have been designed and fabricated, [15][16][17] enabling precise immunomodulation.Such defined molecular patterns (number and geometry) of immuno-components lead to precise interactions between DNA assemblies and immune cells, which is essential for the down-regulation of intracellular signal transduction.Not only inducing a more robust Bcell or T-cell triggering, the reported origami-based artificial APCs even elicited potent tumor growth inhibition at the animal level, [17] revealing novel strategies to drive specific and precise immunomodulatory functions.
Intense investigation of DNA-origami-based nanomaterials is still needed to elucidate their pharmacological performance at the animal level, including in vivo integrity and stability, pharmacokinetics/pharmacodynamics (PK/PD), systemic distribution, metabolism, and clearance.Due to the negative charges of the DNA phosphate backbone, proteins in the blood circulation may adsorb onto the surfaces of the DNA nanostructures, affecting their integrity and in vivo fate.The most commonly used approaches to enhance the in vivo stability of DNA/RNA strands are chemical modifications, among which phosphorothioate linkages and 2'O-methyl ribose modifications have been utilized in clinically approved nucleic acid drugs. [63]With chemical modifications, the circulation time may be further enhanced to a large extent.In addition, delivery platforms on the nanoscale usually exhibit prolonged circulation compared to the corresponding, unencapsulated drugs. [64]Poly(ethylene glycol) (PEG)-coated liposomes showed a significant increase in blood circulation halflife (t 1/2 ≈ 5 h) compared to liposomes without PEG layers (t 1/2 < 30 min), effectively improving the pharmacokinetic profile of drugs and enhancing their bioavailability. [65]For DNA-based delivery systems, tetrahedron structures (≈10 nm) displayed an extended blood circulation time (t 1/2 ≈24.2 min) compared to naked siRNA molecules (t 1/2 ≈ 6 min). [66]For the biodistribution studies, recent advances showed that naked DNA origami nanostructures can accumulate preferentially in the kidneys of mice, exhibiting renal-protective features with similar efficacy to an approved antioxidant agent (N-acetylcysteine) in acute kidney injury (AKI) models. [67]Using these properties of DNA origami structures, a sequential therapeutic strategy for AKI was developed. [68]ectangular DNA origami nanostructures with high renal targeting and long renal retention (>12 h) demonstrated potent antioxidant capacity and effectively alleviated oxidative stress by scavenging ROS during stage I of renal ischemia-reperfusion injury.Subsequently, anticomplement component 5a (aC5a) aptamers loaded on the origami surfaces showed competitive binding and blockade of complement component 5a (C5a), inhibiting inflammatory response in stage II after injury.Additionally, block polymer coverage approaches using PEG or PEG-poly(L-lysine) (PEG-lysine) have been developed to enhance the structural integrity of DNA materials in the biological environment and prolong the half-life in blood circulation. [69]Though the stability of DNA-based material can be improved by polymer coating, these shielding polymers may impact the interaction of DNA materials with biofluids and target tissues/cells.The thickness and density of the polymer covering on the surfaces of the DNA nanostructure may affect the recognition of ligand-receptor pairs or hinder the release of therapeutic cargos from the DNA nanocarriers in the targeted, diseased regions.
Improving targeting strategies to ensure the specific delivery of DNA-origami-based nanocarriers is another area that requires more in-depth study.Currently, clinically approved nucleic acid therapeutics and their corresponding delivery platforms, such as lipid nanoparticle (LNP) formulations and N-acetylgalactosamine (GalNAc) conjugates, preferentially distribute to the liver after administration, leading to low efficiency of encapsulated drug delivery to non-liver tissues.Thus, scientists are developing new methods of precise drug delivery beyond the liver.Alternatively, self-assembled DNA origami nanostructures (ranging from ≈30 to 400 nm) with well-formed geometries can be taken up by and accumulate in tumor regions that exhibit the enhanced permeability and retention (EPR) effect associated with malignant growth. [70]DNA nanostructures can be facilely decorated with multiple moieties, including small molecular ligands, DNA/RNA aptamers, peptides, antibodies, and nanoparticles that are powered by internal/external stimuli, to enhance targeting capacity and specificity.These targeting ligands can be easily located at the desired positions on the addressable surfaces of origami templates, on which the types, numbers, and inter-ligand spacings can be precisely controlled.Besides targeting modifications, the administration route of DNA nanocarriers also affects their biodistribution and tissue accumulation.Rather than via tail vein injection, a DNA nanodevice-based vaccine administered via subcutaneous injection demonstrated optimal migration and retention in lymph nodes, where the loaded therapeutic components were revealed and released, triggering robust immune cell activation and the subsequent priming of systemic anticancer responses. [56]In addition, logic-based DNA nanodevices integrated with specific recognizing/responding motifs can be engineered for disease cell discrimination [71] and weak biological signal amplification. [38]In the future, intelligent systems designed to meet specific biomedical needs are expected to be even more advanced: integrated with multiple ligands and administrated through a specific route.DNA robots/machines may sense and target malignant tissues/cells autonomously, then reconfigure in response to specific biomarkers to release encapsulated drugs in a controlled manner.
As nanoscale delivery strategies continue to improve, innovative DNA-based nanomaterials may benefit from properties shared among the current, clinically relevant delivery systems.For instance, clinical delivery systems tend to be amenable to large-scale fabrication and can be manufactured using chemically simple procedures.As starting materials of DNA origami nanostructures, long scaffold ssDNA molecules and short staples with arbitrary sequences can be generated from high-celldensity fermentation and bacteriophage proliferation in glass or stainless-steel bioreactors, by which upscaled production of gram-amounts of DNA strands has been achieved. [61]With the advances in biotechnological production, the cost of long scaffold DNA can be greatly reduced to US$200 per gram. [61,72]Also, based on the chemically manufactured synthetic strategy, staple strands for DNA origami nanostructures are available for less than US$300 per gram. [73]The versatility of biological and chemical approaches and the reduced cost of raw materials for the production of ssDNA molecules have led to a great leap toward the clinical application of DNA origami.
Currently, approved therapeutic oligonucleotides are simplestructured, single-stranded/double-stranded nucleic acid molecules that are typically manufactured by automated sequential chemical synthesis followed by chromatographic purification.When submitting the initial investigational new drug (IND) and clinical trial application (CTA), all the manufacturing and quality aspects of drug substances and drug products need to be described in the regulatory documents.For instance, impurities in these approved therapeutic oligonucleotides or drug candidates that may originate from raw materials, the synthesis process, degradation during processing, and storage should be carefully addressed and controlled. [74]In contrast, a classic DNA-origami-based nanostructure is constructed by a scaffold strand and hundreds of staple strands through a relatively complex self-assembling process.Good manufacturing practices (GMP)-compliant production of these DNA strands as drug substances that allows for the controlled quality and purity with batch-to-batch consistency would lead to a significant increase in the price. [75]With respect to future clinical translation, the production of DNA-based nanocarriers must be simplified and optimized to enable standard and economical manufacturing processes and quality control procedures.The storage and transportation of nanoformulations are other important considerations.It has been found that the addition of cryoprotectants, such as glycerol and trehalose, at concentrations between 0.2 × 10 −3 and 200 × 10 −3 m, protects DNA origami structures against freeze damage, [76] enabling long-term storage stability.Besides those mentioned issues, non-clinical studies of DNA-based materials must be performed prior to filing an IND application, and most of these assessments need to follow good laboratory practice (GLP) regulations.As novel drug vehicles or candidates move into translational studies, the acceptable PK should be validated and described with stable bioanalytical methods.The therapeutic efficacy/activity of these DNA materials needs to be demonstrated in vitro and in vivo.Dose range finding (DRF) toxicity should be evaluated in-depth at the rodent level or the non-human primate level, which is required for the submission of an IND application.
As a class of promising nucleic-acid-based therapeutics, messenger RNA (mRNA) vaccines have been designed against infectious diseases and several types of cancer. [77]In various preclinical studies and clinical trials, mRNA vaccines have demonstrated therapeutic efficacy and safety, and three coronavirus 2019 (COVID-19) mRNA vaccines (Pfizer-BioNTech and Moderna in the United States, CSPC Pharmaceutical Group in China) have been approved or authorized.6a] Unlike oligonucleotides, mRNA therapeutics are biologically manufactured by in vitro transcription (IVT).Nucleoside-modified mR-NAs have been used in approved vaccines to avoid unintended immune responses.Nanoscale LNP formulations consist of four basic lipid components are common vehicles used for mRNA vaccine delivery. [78]It is worth noting that both the IVT mR-NAs and the scaffold DNA strands for assembling DNA origami are large single-stranded polynucleotides generated by biological manufacturing.A fast and cost-effective purification method of IVT mRNA by cellulose-based chromatography may provide an option for large-scale production of highly pure scaffold DNA.Although LNPs are the most clinically advanced and widely used platforms for mRNA and oligonucleotide delivery, non-LNP vehicles are still needed for in vivo delivery beyond liver and to improve clinical efficacy.Investigation of the successful application of mRNA engineering and delivery as nanomedicine may provide a possible pathway from basic research to clinical translation of DNA nanotechnology.The recent, rapid developments in DNA nanotechnology present great potential for designing new therapeutic strategies.We envision those nucleic-acid-based materials that are programmable, multifunctional, and biologically amenable will provide powerful platforms to better understand and treat disease safely and effectively.

Figure 1 .
Figure 1.DNA origami: an innovative, new class of drug carrier.A long single-stranded DNA scaffold (typically M13mp18 bacteriophage genomic DNA) is folded into an arbitrary origami shape with the aid of multiple, short staple DNA strands.As a candidate for innovative nanocarrier, DNA origami holds distinct advantages, e.g., inherent biodegradability and biocompatibility, arbitrary and high-yield geometries, tailored functionality, and fine-tuned structural reconfiguration.Versatile functional DNA origami nanostructures loaded with active pharmaceutical ingredients (APIs) have been designed and constructed.With their advantageous structural properties, DNA origami technology has yielded various promising platforms for optimized cargo delivery and disease therapy.

Figure 2 .
Figure2.Static DNA origami template based functional assemblies.A) Caspase-9-bound DNA origami as an artificial apoptosome.B) Antigen molecules displayed on a DNA framework for B-cell activation.C) pMHC multimers on triangular DNA origami for T cell regulation and detection.D) Thrombinbinding aptamer nanoarrays on DNA origami for controllable coagulation.E) A genetically encoded DNA origami for gene therapy.F) DNA-origamienabled liposomes for the study of protein-mediated lipid transfer.A) Adapted with permission.[14]Copyright 2020, The Authors, published by Springer Nature.B) Adapted with permission.[15]Copyright 2020, The Authors, published by Springer Nature.C) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0).[16]Copyright 2022, The Authors, published by Springer Nature.D) Adapted under the terms of the CC-BY Creative Commons Attribution license (https://creativecommons.org/licenses/by/4.0).[19]Copyright 2022, The Authors, published by Springer Nature.E) Adapted with permission.[22]Copyright 2023, American Chemical Society.F) Adapted with permission.[31]Copyright 2019, The Authors, published by Springer Nature.

Figure 4 .
Figure 4. Intelligent drug delivery systems based on DNA origami.A) A DNA nanorobot for molecular payload delivery to the surface of a specific cell and manipulation of cell signaling.B) A DNA nanorobot for in vivo thrombin delivery for the precise occlusion of tumor vessels.C) An antigen/adjuvantcodelivery DNA nanodevice vaccine for cancer immunotherapy.A) Adapted with permission.[51]Copyright 2012, The American Association for the Advancement of Science.B) Adapted with permission.[54]Copyright 2018, Springer Nature.C) Adapted with permission.[56]Copyright 2020, The Authors, published by Springer Nature[

Figure 5 .
Figure 5. Challenges facing DNA-based materials as novel drug vehicles.The obstacles, including potential immunogenicity, in vivo stability, targeting strategy, and clinical future, to the widespread application of DNA origami nanocarriers, have been discussed.

Qiao
Jiang received her Bachelor's degree and Master's degree from Xi'an Jiaotong University in 2007 and 2009.She obtained her Ph.D. in 2014 from the National Center for Nanoscience and Technology (NCNST) under the supervision of Professor Baoquan Ding.Following a period as a postdoctoral research fellow in the laboratories at the Institute of Chemistry, Chinese Academy of Sciences, she joined the National Center for Nanoscience and Technology as an assistant professor in 2016.In 2018, she became an associate professor in the NCNST.Her research interests include the design of functional DNA nanoassemblies and drug delivery.