RESUMEN
Technological breakthroughs in cryo-electron microscopy (cryo-EM) methods open new perspectives for highly detailed structural characterizations of extracellular vesicles (EVs) and synthetic liposome-protein assemblies. Structural characterizations of these vesicles in solution under a nearly native hydrated state are of great importance to decipher cell-to-cell communication and to improve EVs' application as markers in diagnosis and as drug carriers in disease therapy. However, difficulties in preparing holey carbon cryo-EM grids with low vesicle heterogeneities, at low concentration and with kinetic control of the chemical reactions or assembly processes, have limited cryo-EM use in the EV study. We report a straightforward membrane vesicle cryo-EM sample preparation method that assists in circumventing these limitations by using a free-standing DNA-affinity superlattice for covering holey carbon cryo-EM grids. Our approach uses DNA origami to self-assemble to a solution-stable and micrometer-sized ordered molecular template in which structure and functional properties can be rationally controlled. We engineered the template with cholesterol-binding sites to specifically trap membrane vesicles. The advantages of this DNA-cholesterol-affinity lattice (DCAL) include (1) local enrichment of artificial and biological vesicles at low concentration and (2) isolation of heterogeneous cell-derived membrane vesicles (exosomes) from a prepurified pellet of cell culture conditioned medium on the grid.
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Microscopía por Crioelectrón , ADN , Microscopía por Crioelectrón/métodos , ADN/química , Vesículas Extracelulares/química , Humanos , Colesterol/química , Liposomas/químicaRESUMEN
Many experimental and computational efforts have sought to understand DNA origami folding, but the time and length scales of this process pose significant challenges. Here, we present a mesoscopic model that uses a switchable force field to capture the behavior of single- and double-stranded DNA motifs and transitions between them, allowing us to simulate the folding of DNA origami up to several kilobases in size. Brownian dynamics simulations of small structures reveal a hierarchical folding process involving zipping into a partially folded precursor followed by crystallization into the final structure. We elucidate the effects of various design choices on folding order and kinetics. Larger structures are found to exhibit heterogeneous staple incorporation kinetics and frequent trapping in metastable states, as opposed to more accessible structures which exhibit first-order kinetics and virtually defect-free folding. This model opens an avenue to better understand and design DNA nanostructures for improved yield and folding performance.
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Nanoestructuras , Nanotecnología , Conformación de Ácido Nucleico , ADN/química , Nanoestructuras/química , CinéticaRESUMEN
Biomolecule-functionalized nanoparticles represent a type of promising biomaterials in biomedical applications owing to their excellent biocompatibility and versatility. DNA-based reactions on nanoparticles have enabled emerging applications including intelligent biosensors, drug delivery, and biomimetic devices. Among the reactions, strand hybridization is the critical step to control the sensitivity and specificity of biosensing, and the efficiency of drug delivery. However, a comprehensive understanding of DNA hybridization on nanoparticles is still lacking, which may differ from the process in homogeneous solutions. To address this limitation, coarse-grained model-based molecular dynamic simulation is harnessed to disclose the critical factors involved in intermolecular hybridization. Based on simulation guidance, DNA walker-based smart theranostic platform (DWTP) based on "on-particle" hybridization is developed, showing excellent consistency with simulation. DWTP is successfully applied for highly sensitive miRNA 21 detection and tumor-specific miRNA 21 imaging, driven by tumor-endogenous APE 1 enzyme. It enables the precise release of antisense oligonucleotide triggered by tumor-endogenous dual-switch miRNA 21 and APE 1, facilitating effective gene silencing therapy with high biosafety. The simulation of "on-particle" DNA hybridization has improved the corresponding biosensing performance and the release efficiency of therapeutic agents, representing a conceptually new approach for DNA-based device design.
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ADN , MicroARNs , Nanomedicina Teranóstica , ADN/química , Nanomedicina Teranóstica/métodos , Humanos , Hibridación de Ácido Nucleico , Nanopartículas/química , Simulación de Dinámica Molecular , Técnicas Biosensibles/métodosRESUMEN
The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.
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Nanoestructuras , Nanoestructuras/química , ADN/química , Oligonucleótidos , Polímeros , Conformación de Ácido Nucleico , NanotecnologíaRESUMEN
Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.
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Técnicas Biosensibles , ADN Catalítico , MicroARNs , ADN Catalítico/metabolismo , ARN Viral , Endonucleasas , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The combination of DNA nanotechnology and Nano Gold (NG) plasmon has opened exciting possibilities for a new generation of functional plasmonic systems that exhibit tailored optical properties and find utility in various applications. In this review, the booming development of dynamic gold nanostructures are summarized, which are formed by DNA self-assembly using DNA-modified NG, DNA frameworks, and various driving forces. The utilization of bottom-up strategies enables precise control over the assembly of reversible and dynamic aggregations, nano-switcher structures, and robotic nanomachines capable of undergoing on-demand, reversible structural changes that profoundly impact their properties. Benefiting from the vast design possibilities, complete addressability, and sub-10 nm resolution, DNA duplexes, tiles, single-stranded tiles and origami structures serve as excellent platforms for constructing diverse 3D reconfigurable plasmonic nanostructures with tailored optical properties. Leveraging the responsive nature of DNA interactions, the fabrication of dynamic assemblies of NG becomes readily achievable, and environmental stimulation can be harnessed as a driving force for the nanomotors. It is envisioned that intelligent DNA-assembled NG nanodevices will assume increasingly important roles in the realms of biological, biomedical, and nanomechanical studies, opening a new avenue toward exploration and innovation.
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ADN , Oro , Oro/química , ADN/química , Nanotecnología/métodos , Nanopartículas del Metal/química , Nanoestructuras/químicaRESUMEN
DNA origami has been used as biotemplates for growing a range of inorganic materials to create novel organic-inorganic hybrid nanomaterials. Recently, the solution-based silicification of DNA has been used to grow thin silica shells on DNA origami. However, the silicification reaction is sensitive to the reaction conditions and often results in uncontrolled DNA origami aggregation, especially when growth of thicker silica layers is desired. Here, we investigated how site-specifically placed polynucleotide brushes influence the silicification of DNA origami. Our experiments showed that long DNA brushes, in the form of single- or double-stranded DNA, significantly suppress the aggregation of DNA origami during the silicification process. Furthermore, we found that double-stranded DNA brushes selectively promote silica growth on DNA origami surfaces. These observations were supported and explained by coarse-grained molecular dynamics simulations. This work provides new insights into our understanding of the silicification process on DNA and provides a powerful toolset for the development of novel DNA-based organic-inorganic nanomaterials.
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Nanoestructuras , Polinucleótidos , Conformación de Ácido Nucleico , ADN , Dióxido de SilicioRESUMEN
Molecular forces generated by cell receptors are infrequent and transient, and hence difficult to detect. Here we report an assay that leverages the CRISPR-associated protein 12a (Cas12a) to amplify the detection of cellular traction forces generated by as few as 50 adherent cells. The assay involves the immobilization of a DNA duplex modified with a ligand specific for a cell receptor. Traction forces of tens of piconewtons trigger the dehybridization of the duplex, exposing a cryptic Cas12-activating strand that sets off the indiscriminate Cas12-mediated cleavage of a fluorogenic reporter strand. We used the assay to perform hundreds of force measurements using human platelets from a single blood draw to extract individualized dose-response curves and half-maximal inhibitory concentrations for a panel of antiplatelet drugs. For seven patients who had undergone cardiopulmonary bypass, platelet dysfunction strongly correlated with the need for platelet transfusion to limit bleeding. The Cas12a-mediated detection of cellular traction forces may be used to assess cell state, and to screen for genes, cell-adhesion ligands, drugs or metabolites that modulate cell mechanics.
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Sistemas CRISPR-Cas , Tracción , Humanos , Adhesión Celular/fisiología , Proteínas , Proteínas PortadorasRESUMEN
Synthetic gene networks in mammalian cells are currently limited to either protein-based transcription factors or RNA-based regulators. Here, we demonstrate a regulatory approach based on circular single-stranded DNA (Css DNA), which can be used as an efficient expression vector with switchable activity, enabling gene regulation in mammalian cells. The Css DNA is transformed into its double-stranded form via DNA replication and used as vectors encoding a variety of different proteins in a wide range of cell lines as well as in mice. The rich repository of DNA nanotechnology allows to use sort single-stranded DNA effectors to fold Css DNA into DNA nanostructures of different complexity, leading the gene expression to programmable inhibition and subsequently re-activation via toehold-mediated strand displacement. The regulatory strategy from Css DNA can thus expand the molecular toolbox for the realization of synthetic regulatory networks with potential applications in genetic diagnosis and gene therapy.
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ADN de Cadena Simple , ADN , Animales , Ratones , ADN de Cadena Simple/genética , ADN/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Mamíferos/genéticaRESUMEN
DNA nanotechnology provides an approach to create precise, tunable, and biocompatible nanostructures for biomedical applications. However, the stability of these structures is severely compromised in biological milieu due to their fast degradation by nucleases. Recently, we showed how enzymatic polymerization could be harnessed to grow polynucleotide brushes of tunable length and location on the surface of DNA origami nanostructures, which greatly enhances their nuclease stability. Here, we report on strategies that allow for both spatial and temporal control over polymerization through activatable initiation, cleavage, and regeneration of polynucleotide brushes using restriction enzymes. The ability to site-specifically decorate DNA origami nanostructures with polynucleotide brushes in a spatiotemporally controlled way provides access to "smart" functionalized DNA architectures with potential applications in drug delivery and supramolecular assembly.
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Nanoestructuras , Polinucleótidos , Nanoestructuras/química , ADN/química , Nanotecnología , Sistemas de Liberación de Medicamentos , Conformación de Ácido NucleicoRESUMEN
Programmable assembly of gold nanoparticle superstructures with precise spatial arrangement has drawn much attention for their unique characteristics in plasmonics and biomedicine. Bio-inspired methods have already provided programmable, molecular approaches to direct AuNP assemblies using biopolymers. The existing methods, however, predominantly use DNA as scaffolds to directly guide the AuNP interactions to produce intended superstructures. New paradigms for regulating AuNP assembly will greatly enrich the toolbox for DNA-directed AuNP manipulation and fabrication. Here, we developed a strategy of using a spatially programmable enzymatic nanorobot arm to modulate anisotropic DNA surface modifications and assembly of AuNPs. Through spatial controls of the proximity of the reactants, the locations of the modifications were precisely regulated. We demonstrated the control of the modifications on a single 15â nm AuNP, as well as on a rectangular DNA origami platform, to direct unique anisotropic AuNP assemblies. This method adds an alternative enzymatic manipulation to DNA-directed AuNP superstructure assembly.
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Structural DNA nanotechnology is capable of fabricating designer nanoscale artificial architectures. Developing simple and yet versatile assembly methods to construct large DNA structures of defined spatial features and dynamic capabilities has remained challenging. Herein, we designed a molecular assembly system where DNA tiles can assemble into tubes and then into large one-dimensional DNA bundles following a hierarchical pathway. A cohesive link was incorporated into the tile to induce intertube binding for the formation of DNA bundles. DNA bundles with length of dozens of micrometers and width of hundreds of nanometers were produced, whose assembly was revealed to be collectively determined by cationic strength and linker designs (binding strength, spacer length, linker position, etc.). Furthermore, multicomponent DNA bundles with programmable spatial features and compositions were realized by using various distinct tile designs. Lastly, we implemented dynamic capability into large DNA bundles to realize reversible reconfigurations among tile, tube, and bundles following specific molecular stimulations. We envision this assembly strategy can enrich the toolbox of DNA nanotechnology for rational design of large-size DNA materials of defined features and properties that may be applied to a variety of fields in materials science, synthetic biology, biomedical science, and beyond.
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ADN , Nanoestructuras , ADN/química , Nanotecnología/métodos , Conformación de Ácido Nucleico , Nanoestructuras/químicaRESUMEN
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.
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Nanoestructuras , ADN , Nanotecnología/métodosRESUMEN
Exploring the structural and electrical properties of DNA origami nanowires is an important endeavor for the advancement of DNA nanotechnology and DNA nanoelectronics. Highly conductive DNA origami nanowires are a desirable target for creating low-cost self-assembled nanoelectronic devices and circuits. In this work, the structure-dependent electrical conductance of DNA origami nanowires is investigated. A silicon nitride (Si3 N4 ) on silicon semiconductor chip with gold electrodes was used for collecting electrical conductance measurements of DNA origami nanowires, which are found to be an order of magnitude less electrically resistive on Si3 N4 substrates treated with a monolayer of hexamethyldisilazane (HMDS) (â¼1013 ohms) than on native Si3 N4 substrates without HMDS (â¼1014 ohms). Atomic force microscopy (AFM) measurements of the height of DNA origami nanowires on mica and Si3 N4 substrates reveal that DNA origami nanowires are â¼1.6â nm taller on HMDS-treated substrates than on the untreated ones indicating that the DNA origami nanowires undergo increased structural deformation when deposited onto untreated substrates, causing a decrease in electrical conductivity. This study highlights the importance of understanding and controlling the interface conditions that affect the structure of DNA and thereby affect the electrical conductance of DNA origami nanowires.
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Nanocables , Nanocables/química , ADN/química , Nanotecnología , Conductividad Eléctrica , Microscopía de Fuerza AtómicaRESUMEN
Antigen recognition by the T cell receptor (TCR) of CD4+ T cells can be greatly enhanced by the coreceptor CD4. Yet, understanding of the molecular mechanism is hindered by the ultra-low affinity of CD4 binding to class-II peptide-major histocompatibility complexes (pMHC). Here we show, using two-dimensional (2D) mechanical-based assays, that the affinity of CD4-pMHC interaction is 3-4 logs lower than that of cognate TCR-pMHC interactions, and it is more susceptible to increased dissociation by forces (slip bond). In contrast, CD4 binds TCR-pre-bound pMHC at 3-6 logs higher affinity, forming TCR-pMHC-CD4 tri-molecular bonds that are prolonged by force (catch bond), and modulated by protein mobility on the cell membrane, indicating profound TCR-CD4 cooperativity. Consistent with a tri-crystal structure, using DNA origami as a molecular ruler to titrate spacing between TCR and CD4 we show that 7-nm proximity optimizes TCR-pMHC-CD4 tri-molecular bond formation with pMHC. Our results thus provide deep mechanistic insight into CD4 enhancement of TCR antigen recognition.
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Antígenos , Receptores de Antígenos de Linfocitos T , Receptores de Antígenos de Linfocitos T/metabolismo , Complejo Mayor de Histocompatibilidad , Antígenos de Histocompatibilidad , Péptidos/químicaRESUMEN
Molecular systems with ability to controllably transform between different conformations play pivotal roles in regulating biochemical functions. Here, we report the design of a bistable DNA origami four-way junction (DOJ) molecular system that adopts two distinct stable conformations with controllable reconfigurability by using conformation-controlled base stacking. Exquisite control over DOJ's conformation and transformation is realized by programming the stacking bonds (quasi-blunt-ends) within the junction to induce prescribed coaxial stacking of neighboring junction arms. A specific DOJ conformation may be achieved by encoding the stacking bonds with binary stacking sequences based on thermodynamic calculations. Dynamic transformations of DOJ between various conformations are achieved by using specific environmental and molecular stimulations to reprogram the stacking codes. This work provides a useful platform for constructing self-assembled DNA nanostructures and nanomachines and insights for future design of artificial molecular systems with increasing complexity and reconfigurability.
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DNA nanostructure-based responsive drug delivery has become an increasingly potent method in cancer therapy. However, a variety of important cancer biomarkers have not been explored in searching of new and efficient targeted delivery systems. Uracil degradation glycosylase and human apurinic/apyrimidinic endonuclease are significantly more active in cancer cells. Here, we developed uracil-modified DNA nanotubes that can deliver drugs to tumor cells through an enzyme-induced disassembly process. Although the reaction of these enzymes on their natural DNA substrates has been established, their reactivity on self-assembled nanostructures of nucleic acids is not well understood. We leveraged molecular dynamic simulation based on coarse-grained model to forecast the enzyme reactivity on different DNA designs. The experimental data are highly consistent with the simulation results. It is the first example of molecule simulation being used to guide the design of enzyme-responsive DNA nano-delivery systems. Importantly, we found that the efficiency of drug release from the nanotubes can be regulated by tuning the positions of uracil modification. The DNA nanotubes equipped with cancer-specific aptamer AS1411 are used to deliver doxorubicin to tumor-bearing mice not only effectively inhibiting tumor growth but also protecting major organs from drug-caused damage. We believe that this work provides new knowledge on and insights into future design of enzyme-responsive DNA-based nanocarriers for drug delivery.
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Nanotubos , Uracil-ADN Glicosidasa , Animales , ADN/química , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Liberación de Fármacos , Humanos , Ratones , Uracilo/metabolismoRESUMEN
Programmable DNA nanotechnology has generated some of the most intricate self-assembled nanostructures and has been employed in a growing number of applications, including functional nanomaterials, nanofabrication, biophysics, photonics, molecular machines, and drug delivery. An important design rule for DNA nanostructures is to minimize the mechanical stress to reduce the potential energy in these nanostructures whenever it is possible. This work revisits the DNA gridiron design consisting of Holliday junctions and compares the self-assembly of the canonical DNA gridiron with a new design of DNA gridiron, which has a higher degree of mechanical stress because of the interweaving of DNA helices. While the interweaving DNA gridiron indeed exhibits lower yield, compared to its canonical counterpart of a similar size, we discover that the mechanical stress within the interweaving gridiron can promote the formation of the two-dimensional crystalline lattice instead of nanotubes. Furthermore, tuning the design of interweaving gridiron leads to the change of overall crystal size and regularity of geometry. Interweaving DNA double helices represents a new design strategy in the self-assembly of DNA nanostructures. Furthermore, the discovery of the new role of mechanical stress in the self-assembly of DNA nanostructures provides useful knowledge to DNA nanotechnology practitioners: a more balanced view regarding mechanical stress can be considered when designing future DNA nanostructures.
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Nanoestructuras , Nanotubos , ADN/química , Nanoestructuras/química , Nanotecnología/métodos , Conformación de Ácido NucleicoRESUMEN
The bacterial flagellar motor is a rotary machine composed of functional modular components, which can perform bidirectional rotations to control the migration behavior of the bacterial cell. It resembles a two-cogwheel gear system, which consists of small and large cogwheels with cogs at the edges to regulate rotations. Such gearset models provide elegant blueprints to design and build artificial nanomachinery with desired functionalities. In this work, we demonstrate DNA assembly of a structurally well-defined nanodevice, which can carry out programmable rotations powered by DNA fuels. Our rotary nanodevice consists of three modular components, small origami ring, large origami ring, and gold nanoparticles (AuNPs). They mimic the sun gear, ring gear, and planet gears in a planetary gearset accordingly. These modular components are self-assembled in a compact manner, such that they can work cooperatively to impart bidirectional rotations. The rotary dynamics is optically recorded using fluorescence spectroscopy in real time, given the sensitive distance-dependent interactions between the tethered fluorophores and AuNPs on the rings. The experimental results are well supported by the theoretical calculations.
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Oro , Nanopartículas del Metal , Oro/química , Nanopartículas del Metal/química , ADN/químicaRESUMEN
Structure-based molecular regulations have been widely adopted to modulate protein networks in cells and recently developed to control allosteric DNA operations in vitro. However, current examples of programmable allosteric signal transmission through integrated DNA networks are stringently constrained by specific design requirements. Developing a new, more general, and programmable scheme for establishing allosteric DNA networks remains challenging. Here, we developed a general strategy for programmable allosteric DNA regulations that can be finely tuned by varying the dimensions, positions, and number of conformational signals. By programming the allosteric signals, we realized fan-out/fan-in DNA gates and multiple-layer DNA cascading networks, as well as expanding the approach to long-range allosteric signal transmission through tunable DNA origami nanomachines ~100 nm in size. This strategy will enable programmable and complex allosteric DNA networks and nanodevices for nanoengineering, chemical, and biomedical applications displaying sense-compute-actuate molecular functionalities.