Soluble and multivalent Jag1 DNA origami nanopatterns activate Notch without pulling force.

The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.

Computer-Aided Design of A-Trail Routed Wireframe DNA Nanostructures with Square Lattice Edges.

In recent years, interest in wireframe DNA origami has increased, with different designs, software, and applications emerging at a fast pace. It is now possible to design a wide variety of shapes by starting with a 2D or 3D mesh and using different scaffold routing strategies. The design choices of the edges in wireframe structures can be important in some applications and have already been shown to influence the interactions between nanostructures and cells. In this work, we increase the alternatives for the design of A-trail routed wireframe DNA structures by using four-helix bundles (4HB). Our approach is based on the incorporation of additional helices to the edges of the wireframe structure to create a 4HB on a square lattice. We first developed the software for the design of these structures, followed by a demonstration of the successful design and folding of a library of structures, and then, finally, we investigated the higher mechanical rigidity of the reinforced structures. In addition, the routing of the scaffold allows us to easily incorporate these reinforced edges together with more flexible, single helix edges, thereby allowing the user to customize the desired stiffness of the structure. We demonstrated the successful folding of this type of hybrid structure and the different stiffnesses of the different parts of the nanostructures using a combination of computational and experimental techniques.

Solid Phase Synthesis of DNA Nanostructures in Heavy Liquid.

Introduction of the solid phase method to synthesize biopolymers has revolutionized the field of biological research by enabling efficient production of peptides and oligonucleotides. One of the advantages of this method is the ease of removal of excess production materials from the desired product, as it is immobilized on solid substrate. The DNA origami method utilizes the nature of nucleotide base-pairing to construct well-defined objects at the nanoscale, and has become a potent tool for manipulating matter in the fields of chemistry, physics, and biology. Here, the development of an approach to synthesize DNA nanostructures directly on magnetic beads, where the reaction is performed in heavy liquid to maintain the beads in suspension is reported. It is demonstrated that the method can achieve high folding yields of up to 90% for various DNA shapes, comparable to standard folding. At the same time, this establishes an easy, fast, and efficient way to further functionalize the DNA origami in one-pot, as well as providing a built-in purification method for easy removal of excess by-products such as non-integrated DNA strands and residual functionalization molecules.

Optical characterization of DNA origami-shaped silver nanoparticles created through biotemplated lithography.

Here, we study optically resonant substrates fabricated using the previously reported BLIN (biotemplated lithography of inorganic nanostructures) technique with single triangle and bowtie DNA origami as templates. We present the first optical characterization of BLIN-fabricated origami-shaped silver nanoparticle patterns on glass surfaces, comprising optical transmission measurements and surface-enhanced Raman spectroscopy. The formed nanoparticle patterns are examined by optical transmission measurements and used for surface enhanced Raman spectroscopy (SERS) of Rhodamine 6G (R6G) dye molecules. Polarization-resolved simulations reveal that the higher SERS enhancement observed for the bowties is primarily due to spectral overlap of the optical resonances with the Raman transitions of R6G. The results manifest the applicability of the BLIN method and substantiate its potential in parallel and high-throughput substrate manufacturing with engineered optical properties. While the results demonstrate the crucial role of the formed nanogaps for SERS, the DNA origami may enable even more complex nanopatterns for various optical applications.

Hybrid Nanoassemblies from Viruses and DNA Nanostructures.

Viruses are among the most intriguing nanostructures found in nature. Their atomically precise shapes and unique biological properties, especially in protecting and transferring genetic information, have enabled a plethora of biomedical applications. On the other hand, structural DNA nanotechnology has recently emerged as a highly useful tool to create programmable nanoscale structures. They can be extended to user defined devices to exhibit a wide range of static, as well as dynamic functions. In this review, we feature the recent development of virus-DNA hybrid materials. Such structures exhibit the best features of both worlds by combining the biological properties of viruses with the highly controlled assembly properties of DNA. We present how the DNA shapes can act as "structured" genomic material and direct the formation of virus capsid proteins or be encapsulated inside symmetrical capsids. Tobacco mosaic virus-DNA hybrids are discussed as the examples of dynamic systems and directed formation of conjugates. Finally, we highlight virus-mimicking approaches based on lipid- and protein-coated DNA structures that may elicit enhanced stability, immunocompatibility and delivery properties. This development also paves the way for DNA-based vaccines as the programmable nano-objects can be used for controlling immune cell activation.

Probing the Conformational States of a pH-Sensitive DNA Origami Zipper via Label-Free Electrochemical Methods.

DNA origami structures represent an exciting class of materials for use in a wide range of biotechnological applications. This study reports the design, production, and characterization of a DNA origami "zipper" structure, which contains nine pH-responsive DNA locks. Each lock consists of two parts that are attached to the zipper's opposite arms: a DNA hairpin and a single-stranded DNA that are able to form a DNA triplex through Hoogsteen base pairing. The sequences of the locks were selected in a way that the zipper adopted a closed configuration at pH 6.5 and an open state at pH 8.0 (transition pKa 7.6). By adding thiol groups, it was possible to immobilize the zipper structure onto gold surfaces. The immobilization process was characterized electrochemically to confirm successful adsorption of the zipper. The open and closed states were then probed using differential pulse voltammetry and electrochemical impedance spectroscopy with solution-based redox agents. It was found that after immobilization, the open or closed state of the zipper in different pH regimes could be determined by electrochemical interrogation. These findings pave the way for development of DNA origami-based pH monitoring and other pH-responsive sensing and release strategies for zipper-functionalized gold surfaces.

Scaling Up DNA Origami Lattice Assembly.

The surface-assisted hierarchical assembly of DNA origami nanostructures is a promising route to fabricate regular nanoscale lattices. In this work, the scalability of this approach is explored and the formation of a homogeneous polycrystalline DNA origami lattice at the mica-electrolyte interface over a total surface area of 18.75 cm2 is demonstrated. The topological analysis of more than 50 individual AFM images recorded at random locations over the sample surface showed only minuscule and random variations in the quality and order of the assembled lattice. The analysis of more than 450 fluorescence microscopy images of a quantum dot-decorated DNA origami lattice further revealed a very homogeneous surface coverage over cm2 areas with only minor boundary effects at the substrate edges. At total DNA costs of €â€…0.12 per cm2 , this large-scale nanopatterning technique holds great promise for the fabrication of functional surfaces.

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release.

Doxorubicin (DOX) is a common drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of DOX-loaded DNA-carriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures (DONs). In our experimental conditions, all DONs show similar DOX binding capacities (one DOX molecule per two to three base pairs), and the binding equilibrium is reached within seconds, remarkably faster than previously acknowledged. To characterize drug release profiles, DON degradation and DOX release from the complexes upon DNase I digestion was studied. For the employed DONs, the relative doses (DOX molecules released per unit time) may vary by two orders of magnitude depending on the DON superstructure. In addition, we identify DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration. These features have been largely ignored in experimenting with DNA nanostructures, but are probably the major sources of the incoherence of the experimental results so far. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA-carriers.

DNA-Origami-Templated Growth of Multilamellar Lipid Assemblies.

Lipids are important building blocks in cellular compartments, and therefore their self-assembly into well-defined hierarchical structures has gained increasing interest. Cationic lipids and unstructured DNA can co-assemble into highly ordered structures (lipoplexes), but potential applications of lipoplexes are still limited. Using scaffolded DNA origami nanostructures could aid in resolving these drawbacks. Here, we have complexed DNA origami together with a cationic lipid 1,2-dioleoly-3-trimethylammonium-propane (DOTAP) and studied their self-assembly driven by electrostatic and hydrophobic interactions. The results suggest that the DNA origami function as templates for the growth of multilamellar lipid structures and that the DNA origami are embedded in the formed lipid matrix. Furthermore, the lipid encapsulation was found to significantly shield the DNA origami against nuclease digestion. The presented complexation strategy is suitable for a wide range of DNA-based templates and could therefore find uses in construction of cell-membrane-associated components.

Robotic DNA Nanostructures.

Over the past decade, DNA nanotechnology has spawned a broad variety of functional nanostructures tailored toward the enabled state at which applications are coming increasingly in view. One of the branches of these applications is in synthetic biology, where the intrinsic programmability of the DNA nanostructures may pave the way for smart task-specific molecular robotics. In brief, the synthesis of the user-defined artificial DNA nano-objects is based on employing DNA molecules with custom lengths and sequences as building materials that predictably assemble together by obeying Watson-Crick base pairing rules. The general workflow of creating DNA nanoshapes is getting more and more straightforward, and some objects can be designed automatically from the top down. The versatile DNA nano-objects can serve as synthetic tools at the interface with biology, for example, in therapeutics and diagnostics as dynamic logic-gated nanopills, light-, pH-, and thermally driven devices. Such diverse apparatuses can also serve as optical polarizers, sensors and capsules, autonomous cargo-sorting robots, rotary machines, precision measurement tools, as well as electric and magnetic-field directed robotic arms. In this review, we summarize the recent progress in robotic DNA nanostructures, mechanics, and their various implementations.

Increasing Complexity in Wireframe DNA Nanostructures.

Structural DNA nanotechnology has recently gained significant momentum, as diverse design tools for producing custom DNA shapes have become more and more accessible to numerous laboratories worldwide. Most commonly, researchers are employing a scaffolded DNA origami technique by "sculpting" a desired shape from a given lattice composed of packed adjacent DNA helices. Albeit relatively straightforward to implement, this approach contains its own apparent restrictions. First, the designs are limited to certain lattice types. Second, the long scaffold strand that runs through the entire structure has to be manually routed. Third, the technique does not support trouble-free fabrication of hollow single-layer structures that may have more favorable features and properties compared to objects with closely packed helices, especially in biological research such as drug delivery. In this focused review, we discuss the recent development of wireframe DNA nanostructures-methods relying on meshing and rendering DNA-that may overcome these obstacles. In addition, we describe each available technique and the possible shapes that can be generated. Overall, the remarkable evolution in wireframe DNA structure design methods has not only induced an increase in their complexity and thus expanded the prevalent shape space, but also already reached a state at which the whole design process of a chosen shape can be carried out automatically. We believe that by combining cost-effective biotechnological mass production of DNA strands with top-down processes that decrease human input in the design procedure to minimum, this progress will lead us to a new era of DNA nanotechnology with potential applications coming increasingly into view.

Advanced DNA Nanopore Technologies.

Diverse nanopore-based technologies have substantially expanded the toolbox for label-free single-molecule sensing and sequencing applications. Biological protein pores, lithographically fabricated solid-state and graphene nanopores, and hybrid pores are in widespread use and have proven to be feasible devices for detecting amino acids, polynucleotides, and their specific conformations. However, despite the indisputable and remarkable advantages in technological exploration and commercialization of such equipment, the commonly used methods may lack modularity and specificity in characterization of particular phenomena or in development of nanopore-based devices. In this review, we discuss DNA nanopore techniques that harness the extreme addressability, precision, and modularity of DNA nanostructures that can be incorporated as customized gates or plugs into for example lipid membranes, solid-state pores, and nanocapillaries, thus forming advanced hybrid instruments. In addition to these, there exist a number of diverse DNA-assisted nanopore-based detection and analysis methods. Here, we introduce different types of DNA nanostructure-based pore designs and their intriguing properties as well as summarize the extensive collection of current and future technologies and applications that can be realized through combining DNA nanotechnology with common nanopore approaches.

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications.

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Real-Time Observation of Superstructure-Dependent DNA Origami Digestion by DNase†I Using High-Speed Atomic Force Microscopy.

DNA nanostructures have emerged as intriguing tools for numerous biomedical applications. However, in many of those applications and most notably in drug delivery, their stability and function may be compromised by the biological media. A particularly important issue for medical applications is their interaction with proteins such as endonucleases, which may degrade the well-defined nanoscale shapes. Herein, fundamental insights into this interaction are provided by monitoring DNase I digestion of four structurally distinct DNA origami nanostructures (DONs) in real time and at a single-structure level by using high-speed atomic force microscopy. The effect of the solid-liquid interface on DON digestion is also assessed by comparison with experiments in bulk solution. It is shown that DON digestion is strongly dependent on its superstructure and flexibility and on the local topology of the individual structure.

Reconfigurable DNA Origami Nanocapsule for pH-Controlled Encapsulation and Display of Cargo.

DNA nanotechnology provides a toolbox for creating custom and precise nanostructures with nanometer-level accuracy. These nano-objects are often static by nature and serve as versatile templates for assembling various molecular components in a user-defined way. In addition to the static structures, the intrinsic programmability of DNA nanostructures allows the design of dynamic devices that can perform predefined tasks when triggered with external stimuli, such as drug delivery vehicles whose cargo display or release can be triggered with a specified physical or chemical cue in the biological environment. Here, we present a DNA origami nanocapsule that can be loaded with cargo and reversibly opened and closed by changing the pH of the surrounding solution. Moreover, the threshold pH value for opening/closing can be rationally designed. We characterize the reversible switching and a rapid opening of "pH-latch"-equipped nanocapsules using Förster resonance energy transfer. Furthermore, we demonstrate the full cycle of capsule loading, encapsulation, and displaying the payload using metal nanoparticles and functional enzymes as cargo mimics at physiologically relevant ion concentrations.

DNA origami directed 3D nanoparticle superlattice via electrostatic assembly.

The arrangements of metal nanoparticles into spatially ordered structures is still challenging, but DNA-based nanostructures have proven to be feasible building blocks in directing the higher-ordered arrangements of nanoparticles. However, an additional DNA functionalization of the particles is often required to link them to the DNA frames. Herein, we show that ordered 3D metal nanoparticle superlattices could be formed also by plainly employing electrostatic interactions between particles and DNA nanostructures. By utilizing the negatively charged DNA origami surface, we were able to assemble 6-helix bundle DNA origami and cationic gold nanoparticles (AuNPs) into well-ordered 3D tetragonal superlattices. Further, the results reveal that shape and charge complementarity between the building blocks are crucial parameters for lattice formation. Our method is not limited to only AuNPs and the origami shapes presented here, and could therefore be used in construction of a variety of functional materials.

Serum Albumin-Peptide Conjugates for Simultaneous Heparin Binding and Detection.

Heparin is a polysaccharide-based anticoagulant agent, which is widely used in surgery and blood transfusion. However, overdosage of heparin may cause severe side effects such as bleeding and low blood platelet count. Currently, there is only one clinically licensed antidote for heparin: protamine sulfate, which is known to provoke adverse effects. In this work, we present a stable and biocompatible alternative for protamine sulfate that is based on serum albumin, which is conjugated with a variable number of heparin-binding peptides. The heparin-binding efficiency of the conjugates was evaluated with methylene blue displacement assay, dynamic light scattering, and anti-Xa assay. We found that multivalency of the peptides played a key role in the observed heparin-binding affinity and complex formation. The conjugates had low cytotoxicity and low hemolytic activity, indicating excellent biocompatibility. Furthermore, a sensitive DNA competition assay for heparin detection was developed. The detection limit of heparin was 0.1 IU/mL, which is well below its therapeutic range (0.2-0.4 IU/mL). Such biomolecule-based systems are urgently needed for next-generation biocompatible materials capable of simultaneous heparin binding and sensing.

DNA Origami Nanophotonics and Plasmonics at Interfaces.

DNA nanotechnology provides a versatile toolbox for creating custom and accurate shapes that can serve as versatile templates for nanopatterning. These DNA templates can be used as molecular-scale precision tools in, for example, biosensing, nanometrology, and super-resolution imaging, and biocompatible scaffolds for arranging other nano-objects, for example, for drug delivery applications and molecular electronics. Recently, increasing attention has been paid to their potent use in nanophotonics since these modular templates allow a wide range of plasmonic and photonic ensembles ranging from DNA-directed nanoparticle and fluorophore arrays to entirely metallic nanostructures. This Feature Article focuses on the DNA-origami-based nanophotonics and plasmonics-especially on the methods that take advantage of various substrates and interfaces for the foreseen applications.

Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems.

DNA nanotechnology provides an excellent foundation for diverse nanoscale structures that can be used in various bioapplications and materials research. Among all existing DNA assembly techniques, DNA origami proves to be the most robust one for creating custom nanoshapes. Since its invention in 2006, building from the bottom up using DNA advanced drastically, and therefore, more and more complex DNA-based systems became accessible. So far, the vast majority of the demonstrated DNA origami frameworks are static by nature; however, there also exist dynamic DNA origami devices that are increasingly coming into view. In this review, we discuss DNA origami nanostructures that exhibit controlled translational or rotational movement when triggered by predefined DNA sequences, various molecular interactions, and/or external stimuli such as light, pH, temperature, and electromagnetic fields. The rapid evolution of such dynamic DNA origami tools will undoubtedly have a significant impact on molecular-scale precision measurements, targeted drug delivery and diagnostics; however, they can also play a role in the development of optical/plasmonic sensors, nanophotonic devices, and nanorobotics for numerous different tasks.

DNA-Assisted Molecular Lithography.

During the past decade, DNA origami has become a popular method to build custom two- (2D) and three-dimensional (3D) DNA nanostructures. These programmable structures could further serve as templates for accurate nanoscale patterning, and therefore they could find uses in various biotechnological applications. However, to transfer the spatial information of DNA origami to metal nanostructures has been limited to either direct nanoparticle-based patterning or chemical growth of metallic seed particles that are attached to the DNA objects. Here, we present an alternative way by combining DNA origami with conventional lithography techniques. With this DNA-assisted lithography (DALI) method, we can create plasmonic, entirely metallic nanostructures in a highly accurate and parallel manner on different substrates. We demonstrate our technique by patterning a transparent substrate with discrete bowtie-shaped nanoparticles, i.e., "nanoantennas" or "optical antennas," with a feature size of approximately 10 nm. Owing to the versatility of DNA origami, this method can be effortlessly generalized to other shapes and sizes.