Multipathway Regulation for Targeted Atherosclerosis Therapy Using Anti-miR-33-Loaded DNA Origami.

Given the multifactorial pathogenesis of atherosclerosis (AS), a chronic inflammatory disease, combination therapy arises as a compelling approach to effectively address the complex interplay of pathogenic mechanisms for a more desired treatment outcome. Here, we present cRGD/ASOtDON, a nanoformulation based on a self-assembled DNA origami nanostructure for the targeted combination therapy of AS. cRGD/ASOtDON targets αvß3 integrin receptors overexpressed on pro-inflammatory macrophages and activated endothelial cells in atherosclerotic lesions, alleviates the oxidative stress induced by extracellular and endogenous reactive oxygen species, facilitates the polarization of pro-inflammatory macrophages toward the anti-inflammatory M2 phenotype, and inhibits foam cell formation by promoting cholesterol efflux from macrophages by downregulating miR-33. The antiatherosclerotic efficacy and safety profile of cRGD/ASOtDON, as well as its mechanism of action, were validated in an AS mouse model. cRGD/ASOtDON treatment reversed AS progression and restored normal morphology and tissue homeostasis of the diseased artery. Compared to probucol, a clinical antiatherosclerotic drug with a similar mechanism of action, cRGD/ASOtDON enabled the desired therapeutic outcome at a notably lower dosage. This study demonstrates the benefits of targeted combination therapy in AS management and the potential of self-assembled DNA nanoformulations in addressing multifactorial inflammatory conditions.

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

DNA-Modulated and Mechanoresponsive Excitonic Couplings Reveal Chiroptical Correlation of Conformation, Tension, and Dynamics of DNA Self-Assembly.

Study of the conformational and mechanical behaviors of biomolecular assemblies is vital to the rational design and realization of artificial molecular architectures with biologically relevant functionality. Here, we revealed DNA-modulated and mechanoresponsive excitonic couplings between organic chromophores and verified strong correlations between the excitonic chiroptical responses and the conformational and mechanical states of DNA self-assemblies irrespective of fluorescence background interference. Besides, the excitonic chiroptical effect allowed sensitive monitoring of DNA self-assembled nanostructures due to small molecule bindings or DNA strand displacement reactions. Moreover, we developed a new chiroptical reporter, a DNA-templated dimer of an achiral cyanine5 and an intrinsically chiral BODIPY, that exhibited unique multiple-split spectral line shape of exciton-coupled circular dichroism, largely separated response wavelengths, and enhanced anisotropy dissymmetry factor (g-factor). These results shed light on a promising chiroptical spectroscopic tool for studying biomolecular recognition and binding, conformation dynamics, and soft mechanics in general.

CytoDirect: A Nucleic Acid Nanodevice for Specific and Efficient Delivery of Functional Payloads to the Cytoplasm.

Direct and efficient delivery of functional payloads such as chemotherapy drugs, siRNA, or small-molecule inhibitors into the cytoplasm, bypassing the endo/lysosomal trapping, is a challenging task for intracellular medicine. Here, we take advantage of the programmability of DNA nanotechnology to develop a DNA nanodevice called CytoDirect, which incorporates disulfide units and human epidermal growth factor receptor 2 (HER2) affibodies into a DNA origami nanostructure, enabling rapid cytosolic uptake into targeted cancer cells and deep tissue penetration. We further demonstrated that therapeutic oligonucleotides and small-molecule chemotherapy drugs can be easily delivered by CytoDirect and showed notable effects on gene knockdown and cell apoptosis, respectively. This study demonstrates the synergistic effect of disulfide and HER2 affibody modifications on the rapid cytosolic delivery of DNA origami and its payloads to targeted cells and deep tissues, thereby expanding the delivery capabilities of DNA nanostructures in a new direction for disease treatment.

Translocation of Proteins through Solid-State Nanopores Using DNA Polyhedral Carriers.

The detection of biomolecules at the single molecule level has important applications in the fields of biosensing and biomedical diagnosis. The solid-state nanopore (SS nanopore) is a sensitive tool for detecting single molecules because of its unique label-free and low sample consumption properties. SS nanopore translocation of small biomolecules is typically driven by an electronic field force and is thus influenced by the charge, shape, and size of the target molecules. Therefore, it remains challenging to control the translocation of biomolecules through SS nanopores, particularly for different proteins with complex conformations and unique charges. Toward this problem, a DNA polyhedral carrier coating strategy to assist protein translocation through SS nanopores is developed, which facilitates target protein detection. The current signal-to-noise ratios are improved significantly using this DNA carrier loading strategy. The proposed method should aid the detection of proteins, which are difficult to translocate through nanopores. This coating-assisted method offers a wide range of applications for SS nanopore detection and promotes the development of single-molecule detection.

DNA Origami Tessellations.

Molecular tessellation research aims to elucidate the underlying principles that govern intricate patterns in nature and to leverage these principles to create precise and ordered structures across multiple scales, thereby facilitating the emergence of novel functionalities. DNA origami nanostructures are excellent building blocks for constructing tessellation patterns. However, the size and complexity of DNA origami tessellation systems are currently limited by several unexplored factors relevant to the accuracy of essential design parameters, the applicability of design strategies, and the compatibility between different tiles. Here, we present a general method for creating DNA origami tiles that grow into tessellation patterns with micrometer-scale order and nanometer-scale precision. Interhelical distance (D) was identified as a critical design parameter determining tile conformation and tessellation outcome. Finely tuned D facilitated the accurate geometric design of monomer tiles with minimized curvature and improved tessellation capability, enabling the formation of single-crystalline lattices ranging from tens to hundreds of square micrometers. The general applicability of the design method was demonstrated by 9 tile geometries, 15 unique tile designs, and 12 tessellation patterns covering Platonic, Laves, and Archimedean tilings. Particularly, we took two strategies to increase the complexity of DNA origami tessellation, including reducing the symmetry of monomer tiles and coassembling tiles of different geometries. Both yielded various tiling patterns that rivaled Platonic tilings in size and quality, indicating the robustness of the optimized tessellation system. This study will promote DNA-templated, programmable molecular and material patterning and open up new opportunities for applications in metamaterial engineering, nanoelectronics, and nanolithography.

Two-Dimensional Excitonic Networks Directed by DNA Templates as an Efficient Model Light-Harvesting and Energy Transfer System.

Photosynthetic organisms organize discrete light-harvesting complexes into large-scale networks to facilitate efficient light collection and utilization. Inspired by nature, herein, synthetic DNA templates were used to direct the formation of dye aggregates with a cyanine dye, K21, into discrete branched photonic complexes, and two-dimensional (2D) excitonic networks. The DNA templates ranged from four-arm DNA tiles, ≈10 nm in each arm, to 2D wireframe DNA origami nanostructures with different geometries and varying dimensions up to 100×100 nm. These DNA-templated dye aggregates presented strongly coupled spectral features and delocalized exciton characteristics, enabling efficient photon collection and energy transfer. Compared to the discrete branched photonic systems templated on individual DNA tiles, the interconnected excitonic networks showed approximately a 2-fold increase in energy transfer efficiency. This bottom-up assembly strategy paves the way to create 2D excitonic systems with complex geometries and engineered energy pathways.

DNA Origami as a Nanomedicine for Targeted Rheumatoid Arthritis Therapy through Reactive Oxygen Species and Nitric Oxide Scavenging.

Rheumatoid arthritis (RA) severely threatens human health by causing inflammation, swelling, and pain in the joints and resulting in persistent synovitis and irreversible joint disability. In the development of RA, pro-inflammatory M1 macrophages, which express high levels of reactive oxygen species (ROS) and nitric oxide (NO), induce synovial inflammation and bone erosion. Eliminating ROS and NO in the inflamed joints is a potential RA therapeutic approach, which can drive the transition of pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype. Taking advantage of the intrinsic ROS- and NO-scavenging capability of DNA molecules, herein, we report the development of folic acid-modified triangular DNA origami nanostructures (FA-tDONs) for targeted RA treatment. FA-tDONs could efficiently scavenge ROS and NO and actively target M1 macrophages, facilitating the M1-to-M2 transition and the recovery of associated cytokines and biomarkers to the normal level. The therapeutic efficacy of FA-tDONs was examined in the RA mouse model. As validated by appearance, histological, and serum examinations, FA-tDONs treatment effectively alleviated synovial infiltration and cartilage damage, attenuating disease progression. This study demonstrated the usage of DNA origami for RA treatment and suggested its potential in other antioxidant therapies.

DNA-Assembled Chiral Satellite-Core Nanoparticle Superstructures: Two-State Chiral Interactions from Dynamic and Static Conformations.

A significant challenge exists in obtaining chiral nanostructures that are amenable to both solution-phase self-assembly and solid-phase preservation, which enable the observation of unveiled optical responses impacted by the dynamic or static conformation and the incident excitations. Here, to meet this demand, we employed DNA origami technology to create quasi-planar chiral satellite-core nanoparticle superstructures with an intermediate geometry between the monolayer and the double layer. We disentangled the complex chiral mechanisms, which include planar chirality, 3D chirality, and induced chirality transfer, through combined theoretical studies and thorough experimental measurements of both solution- and solid-phase samples. Two distinct states of optical responses were demonstrated by the dynamic and static conformations, involving a split or nonsplit circular dichroism (CD) line shape. More importantly, our study on chiral nanoparticle superstructures on a substrate featuring both a dominant 2D geometry and a defined 3D represents a great leap toward the realization of colloidal chiral metasurfaces.

A localized DNA finite-state machine with temporal resolution.

The identity and timing of environmental stimulus play a pivotal role in living organisms in programming their signaling networks and developing specific phenotypes. The ability to unveil history-dependent signals will advance our understanding of temporally regulated biological processes. Here, we have developed a two-input, five-state DNA finite-state machine (FSM) to sense and record the temporally ordered inputs. The spatial organization of the processing units on DNA origami enables facile modulation of the energy landscape of DNA strand displacement reactions, allowing precise control of the reactions along predefined paths for different input orders. The use of spatial constraints brings about a simple, modular design for the FSM with a minimum set of orthogonal components and confers minimized leaky reactions and fast kinetics. The FSM demonstrates the capability of sensing the temporal orders of two microRNAs, highlighting its potential for temporally resolved biosensing and smart therapeutics.

DNA Programmable Self-Assembly of Planar, Thin-Layered Chiral Nanoparticle Superstructures with Complex Two-Dimensional Patterns.

Planar, thin-layered chiral plasmonic superstructures with complex two-dimensional (2D) patterns, namely, double-layered binary stars (bi-stars) and pinwheels, were realized through DNA programmable 2D supramolecular self-assembly of gold nanorods (AuNRs). The chirality of the chiral superstructures was defined by a finite number of AuNR pairs as enantiomeric motifs, and their sizes (∼240 nm) were precisely defined by the underlying DNA template. These planar, thin-layered chiral nanoparticle superstructures exhibited prescribed shapes and sizes at the dried state on the substrate surface and are characteristic of giant anisotropy of chiroptical responses, with enhanced g-factors from the axial incident excitation as compared to the in-plane excitation. This work will inspire possibilities for the construction of 2D chiral materials, for example, chiral metasurfaces, for the on-chip manipulation of chiral light-matter interactions via programmable self-assembly of nanoparticles.

Regulating DNA Self-Assembly Dynamics with Controlled Nucleation.

Controlling the nucleation step of a self-assembly system is essential for engineering structural complexity and dynamic behaviors. Here, we design a "frame-filling" model system that comprises one type of self-complementary DNA tile and a hosting DNA origami frame to investigate the inherent dynamics of three general nucleation modes in nucleated self-assembly: unseeded, facet, and seeded nucleation. Guided by kinetic simulation, which suggested an optimal temperature range to differentiate the individual nucleation modes, and complemented by single-molecule observations, the transition of tiles from a metastable, monomeric state to a stable, polymerized state through the three nucleation pathways was monitored by Mg2+-triggered kinetic measurements. The temperature-dependent kinetics for all three nucleation modes were correlated by a "nucleation-growth" model, which quantified the tendency of nucleation using an empirical nucleation number. Moreover, taking advantage of the temperature dependence of nucleation, tile assembly can be regulated externally by the hosting frame. An ultraviolet (UV)-responsive trigger was integrated into the frame to simultaneously control "when" and "where" nucleation started. Our results reveal the dynamic mechanisms of the distinct nucleation modes in DNA tile-based self-assembly and provide a general strategy for controlling the self-assembly process.

Meta-DNA structures.

DNA origami has emerged as a highly programmable method to construct customized objects and functional devices in the 10-100 nm scale. Scaling up the size of the DNA origami would enable many potential applications, which include metamaterial construction and surface-based biophysical assays. Here we demonstrate that a six-helix bundle DNA origami nanostructure in the submicrometre scale (meta-DNA) could be used as a magnified analogue of single-stranded DNA, and that two meta-DNAs that contain complementary 'meta-base pairs' can form double helices with programmed handedness and helical pitches. By mimicking the molecular behaviours of DNA strands and their assembly strategies, we used meta-DNA building blocks to form diverse and complex structures on the micrometre scale. Using meta-DNA building blocks, we constructed a series of DNA architectures on a submicrometre-to-micrometre scale, which include meta-multi-arm junctions, three-dimensional (3D) polyhedrons, and various 2D/3D lattices. We also demonstrated a hierarchical strand-displacement reaction on meta-DNA to transfer the dynamic features of DNA into the meta-DNA. This meta-DNA self-assembly concept may transform the microscopic world of structural DNA nanotechnology.

Encoding quantized fluorescence states with fractal DNA frameworks.

Signal amplification in biological systems is achieved by cooperatively recruiting multiple copies of regulatory biomolecules. Nevertheless, the multiplexing capability of artificial fluorescent amplifiers is limited due to the size limit and lack of modularity. Here, we develop Cayley tree-like fractal DNA frameworks to topologically encode the fluorescence states for multiplexed detection of low-abundance targets. Taking advantage of the self-similar topology of Cayley tree, we use only 16 DNA strands to construct n-node (n = 53) structures of up to 5 megadalton. The high level of degeneracy allows encoding 36 colours with 7 nodes by site-specifically anchoring of distinct fluorophores onto a structure. The fractal topology minimises fluorescence crosstalk and allows quantitative decoding of quantized fluorescence states. We demonstrate a spectrum of rigid-yet-flexible super-multiplex structures for encoded fluorescence detection of single-molecule recognition events and multiplexed discrimination of living cells. Thus, the topological engineering approach enriches the toolbox for high-throughput cell imaging.

RNA Origami Nanostructures for Potent and Safe Anticancer Immunotherapy.

Rapid developments in nucleic acid nanotechnology have enabled the rational design and construction of self-assembling DNA and RNA nanostructures that are highly programmable. We recently developed a replicable single-stranded RNA origami (RNA-OG) technology that allows a long RNA molecule to be programmed to self-assemble into nanostructures of various shapes. Here, we show that such RNA-OG is highly stable in serum/plasma, and we thus exploited its immunostimulatory potential. We demonstrated that the RNA-OG stimulates a potent innate response primarily through a Toll-like receptor 3 (TLR3) pathway. In a murine peritoneal metastatic colon cancer model, intraperitoneally injected RNA-OG induced significant tumor retardation or regression by activating NK- and CD8-dependent antitumor immunity and antagonizing the peritoneal immunosuppressive environment. Unlike polyinosinic/polycytidylic acid (PolyIC), a well-known double-stranded RNA analogue, the RNA-OG treatment did not cause a high level of type-I interferons in the blood nor apparent toxicity upon its systemic administration in the animals. This work establishes the function of RNA-OG as a potent line of TLR3 agonists that are safe and effective for cancer immunotherapy.

Efficient Long-Range, Directional Energy Transfer through DNA-Templated Dye Aggregates.

The benzothiazole cyanine dye K21 forms dye aggregates on double-stranded DNA (dsDNA) templates. These aggregates exhibit a red-shifted absorption band, enhanced fluorescence emission, and an increased fluorescence lifetime, all indicating strong excitonic coupling among the dye molecules. K21 aggregate formation on dsDNA is only weakly sequence dependent, providing a flexible approach that is adaptable to many different DNA nanostructures. Donor (D)-bridge (B)-acceptor (A) complexes consisting of Alexa Fluor 350 as the donor, a 30 bp (9.7 nm) DNA templated K21 aggregate as the bridge, and Alexa Fluor 555 as the acceptor show an overall donor to acceptor energy transfer efficiency of ∼60%, with the loss of excitation energy being almost exclusively at the donor-bridge junction (63%). There was almost no excitation energy loss due to transfer through the aggregate bridge, and the transfer efficiency from the aggregate to the acceptor was about 96%. By comparing the energy transfer in templated aggregates at several lengths up to 32 nm, the loss of energy per nanometer through the K21 aggregate bridge was determined to be <1%, suggesting that it should be possible to construct structures that use much longer energy transfer "wires" for light-harvesting applications in photonic systems.

Tunable Nanoscale Cages from Self-Assembling DNA and Protein Building Blocks.

Three-dimensional (3D) cages are one of the most important targets for nanotechnology. Both proteins and DNA have been used as building blocks to create tunable nanoscale cages for a wide range of applications, but each molecular type has its own limitations. Here, we report a cage constructed from both protein and DNA building blocks through the use of covalent protein-DNA conjugates. We modified a homotrimeric protein (KDPG aldolase) with three identical single-stranded DNA handles by functionalizing a reactive cysteine residue introduced via site-directed mutagenesis. This protein-DNA building block was coassembled with a triangular DNA structure bearing three complementary arms to the handles, resulting in tetrahedral cages comprising six DNA sides capped by the protein trimer. The dimensions of the cage could be tuned through the number of turns per DNA arm (3 turns ∼ 10 nm, 4 turns ∼ 14 nm), and the hybrid structures were purified and characterized to confirm the three-dimensional structure. Cages were also modified with DNA using click chemistry and using aldolase trimers bearing the noncanonical amino acid 4-azidophenylalanine, demonstrating the generality of the method. Our approach will allow for the construction of nanomaterials that possess the advantages of both protein and DNA nanotechnology and find applications in fields such as targeted delivery, structural biology, biomedicine, and catalytic materials.