Structural DNA Nanotechnology Research Articles

Probing the Formation Kinetics and Thermodynamics with Rationally Designed Analytical Tools Enables One-Pot Synthesis and Purification of a Tetrahedral DNA Nanostructure.

The development of robust analytical tools capable of probing the formation kinetics and thermodynamics of DNA nanostructures is a crucial step toward better understanding and manufacturing of diverse DNA-based materials. Herein, we introduce a real-time fluorescence anisotropy assay and rationally designed DNA reaction termination probes (DRTPs) as a set of new tools for exploring the formation mechanisms of DNA nanostructures. We deployed these tools for probing the formation of a classic tetrahedral DNA nanostructure (TDN) as a model system. Our tools revealed that the formation of TDN was dominated by simultaneous hybridization, whereas its undesired side products were caused mainly through step-wise hybridization. An optimal reaction temperature exists that favors the formation of TDN over side products. With insight into the TDN formation mechanism, we further engineered magnetic DRTPs to achieve single-step purification of TDN, enabling 10-fold improvement in the ratio between the targeted TDN and undesired side products without tedious procedures or bulky instruments. Combining the optimal reaction and purification conditions, we finally demonstrated the one-pot synthesis and purification of TDN. The analytical techniques offered in this work may hold potential to find wide applications and inspire new analytical methods for structural DNA nanotechnology.

Generating DNA Origami Nanostructures through Shape Annealing

Structural DNA nanotechnology involves the design and self-assembly of DNA-based nanostructures. As a field, it has progressed at an exponential rate over recent years. The demand for unique DNA origami nanostructures has driven the development of design tools, but current CAD tools for structural DNA nanotechnology are limited by requiring users to fully conceptualize a design for implementation. This article introduces a novel formal approach for routing the single-stranded scaffold DNA that defines the shape of DNA origami nanostructures. This approach for automated scaffold routing broadens the design space and generates complex multilayer DNA origami designs in an optimally driven way, based on a set of constraints and desired features. This technique computes unique designs of DNA origami assemblies by utilizing shape annealing, which is an integration of shape grammars and the simulated annealing algorithm. The results presented in this article illustrate the potential of the technique to code desired features into DNA nanostructures.

3D DNA Nanostructures: The Nanoscale Architect

Structural DNA nanotechnology is a pioneering biotechnology that presents the opportunity to engineer DNA-based hardware that will mediate a profound interface to the nanoscale. To date, an enormous library of shaped 3D DNA nanostructures have been designed and assembled. Moreover, recent research has demonstrated DNA nanostructures that are not only static but can exhibit specific dynamic motion. DNA nanostructures have thus garnered significant research interest as a template for pursuing shape and motion-dependent nanoscale phenomena. Potential applications have been explored in many interdisciplinary areas spanning medicine, biosensing, nanofabrication, plasmonics, single-molecule chemistry, and facilitating biophysical studies. In this review, we begin with a brief overview of general and versatile design techniques for 3D DNA nanostructures as well as some techniques and studies that have focused on improving the stability of DNA nanostructures in diverse environments, which is pivotal for its reliable utilization in downstream applications. Our main focus will be to compile a wide body of existing research on applications of 3D DNA nanostructures that demonstrably rely on the versatility of their mechanical design. Furthermore, we frame reviewed applications into three primary categories, namely encapsulation, surface templating, and nanomechanics, that we propose to be archetypal shape- or motion-related functions of DNA nanostructures found in nanoscience applications. Our intent is to identify core concepts that may define and motivate specific directions of progress in this field as we conclude the review with some perspectives on the future.

Hybrid Wireframe DNA Nanostructures with Scaffolded and Scaffold‐Free Modules

AbstractThe scaffolded DNA origami approach and the scaffold‐free LEGO approach both demonstrate an extraordinary self‐assembly capability for constructing all kinds of complex DNA nanostructures. Combining the construction elements of the two approaches, we introduce a hybrid framework to build wireframe structures in this study. A collection of two‐dimensional (2D) and three‐dimensional (3D) wireframe structures are presented to showcase the simple and versatile design. Our development reveals more of the ever‐expanding design space of structural DNA nanotechnology.

Hybrid Wireframe DNA Nanostructures with Scaffolded and Scaffold‐Free Modules

The scaffolded DNA origami approach and the scaffold-free LEGO approach both demonstrate an extraordinary self-assembly capability for constructing all kinds of complex DNA nanostructures. Combining the construction elements of the two approaches, we introduce a hybrid framework to build wireframe structures in this study. A collection of two-dimensional (2D) and three-dimensional (3D) wireframe structures are presented to showcase the simple and versatile design. Our development reveals more of the ever-expanding design space of structural DNA nanotechnology.

Single Molecule Shape Determination of Biomolecules using DNA Nanoswitch Calipers

Single-molecule mechanical measurements can provide an unprecedented view into the shape, stabilities, and dynamics of biomolecules and their interactions. However, current challenges in determining the spatial arrangement of multiple residues within each molecule with a reasonable resolution and accuracy, have restricted applications. By combining structural DNA nanotechnology with dual-trap optical tweezers, we recently developed a mechanically reconfigurable DNA Nanoswitch Caliper (DNC) capable of measuring multiple distances on single biomolecules with atomic resolution.

Structural DNA nanotechnology towards therapeutic applications

A DNA molecule is a genetic material composed of a double helix. Inspired by its unique characteristics, scientists have exploited DNA strands for engineering nanostructures. The development of DNA origami, which has enabled the customized production of DNA nanostructures, has been of crucial significance. Ever since, various DNA nanostructures have been proposed, promising the effective treatment of diverse diseases. In particular, we introduce the key strategies and technological advances of a range of DNA nanostructures, including DNA-based drug delivery systems and DNA nanorobots.

Development and functionalization of DNA nanostructures for biomedical applications

AbstractNanomaterials are excellent drug delivery systems, yet, they must be functionalized in a manner compatible with the biological environment. Regarding delivery of the payloads, it is critical to monitor the nanocarrier's biocompatibility and the ability to control its drug encapsulation and release, as well as targeting. The current challenges include avoiding negative host immune responses, optimizing stability in biological environments, and achieving precise interactions with the targets. Contemporary advances in structural DNA nanotechnology, DNA origami, and supramolecular DNA assembly make it possible to produce complex multi‐functional DNA nanostructures, wherein precise control of the size, geometry, and appearance of the ligands is feasible. DNA nanostructures offer ease of synthesis and conjugation of functional moieties to target the release of cargo or to analyze important biomarkers for diagnostics. Furthermore, the biocompatibility, programmability, responsiveness to biomolecules, cell‐surfaces, and organisms make such DNA nanomaterials highly suitable for potential translational applications. This overview summarizes the recent developments in functionalizing, stabilizing, and applying DNA nanostructures for potential biomedical applications.

Rapid Computational Analysis of DNA Origami Assemblies at Near-Atomic Resolution

Structural DNA nanotechnology plays an ever-increasing role in advanced biomolecular applications. Here, we present a computational method to analyze structured DNA assemblies rapidly at near-atomic resolution. Both high computational efficiency and molecular-level accuracy are achieved by developing a multiscale analysis framework. The sequence-dependent relative geometry and mechanical properties of DNA motifs are characterized by the all-atom molecular dynamics simulation and incorporated into the structural finite element model successfully without significant loss of atomic information. The proposed method can predict the three-dimensional shape, equilibrium dynamic properties, and mechanical rigidities of monomeric to hierarchically assembled DNA structures at near-atomic resolution without adjusting any model parameters. The calculation takes less than only 15 min for most origami-scale DNA nanostructures consisting of 7000-8000 base-pairs. Hence, it is expected to be highly utilized in an iterative design-analysis-revision process for structured DNA assemblies.

The effects of overhang placement and multivalency on cell labeling by DNA origami.

Through targeted binding to the cell membrane, structural DNA nanotechnology has the potential to guide and affix biomolecules such as drugs, growth factors and nanobiosensors to the surfaces of cells. In this study, we investigated the targeted binding efficiency of three distinct DNA origami shapes to cultured endothelial cells via cholesterol anchors. Our results showed that the labeling efficiency is highly dependent on the shape of the origami as well as the number and the location of the binding overhangs. With a uniform surface spacing of binding overhangs, 3D isotropic nanospheres and 1D anisotropic nanorods labeled cells effectively, and the isotropic nanosphere labeling fit well with an independent binding model. Face-decoration and edge-decoration of the anisotropic nanotile were performed to investigate the effects of binding overhang location on cell labeling, and only the edge-decorated nanotiles were successful at labeling cells. Edge proximity studies demonstrated that the labeling efficiency can be modulated in both nanotiles and nanorods by moving the binding overhangs towards the edges and vertices, respectively. Furthermore, we demonstrated that while double-stranded DNA (dsDNA) bridge tethers can rescue the labeling efficiency of the face-decorated rectangular plate, this effect is also dependent on the proximity of bridge tethers to the edges or vertices of the nanostructures. A final comparison of all three nanoshapes revealed that the end-labeled nanorod and the nanosphere achieved the highest absolute labeling intensities, but the highest signal-to-noise ratio, calculated as the ratio of overall labeling to initiator-free background labeling, was achieved by the end-labeled nanorod, with the edge-labeled nanotile coming in second place slightly ahead of the nanosphere. The findings from this study can help us further understand the factors that affect membrane attachment using cholesterol anchors, thus providing guidelines for the rational design of future functional DNA nanostructures.

Design Approaches and Computational Tools for DNA Nanostructures.

Designing a structure in nanoscale with desired shape and properties has been enabled by structural DNA nanotechnology. Design strategies in this research field have evolved to interpret various aspects of increasingly more complex nanoscale assembly and to realize molecular-level functionality by exploring static to dynamic characteristics of the target structure. Computational tools have naturally been of significant interest as they are essential to achieve a fine control over both shape and physicochemical properties of the structure. Here, we review the basic design principles of structural DNA nanotechnology together with its computational analysis and design tools.

Insights into the Structure and Energy of DNA Nanoassemblies.

Since the pioneering work of Ned Seeman in the early 1980s, the use of the DNA molecule as a construction material experienced a rapid growth and led to the establishment of a new field of science, nowadays called structural DNA nanotechnology. Here, the self-recognition properties of DNA are employed to build micrometer-large molecular objects with nanometer-sized features, thus bridging the nano- to the microscopic world in a programmable fashion. Distinct design strategies and experimental procedures have been developed over the years, enabling the realization of extremely sophisticated structures with a level of control that approaches that of natural macromolecular assemblies. Nevertheless, our understanding of the building process, i.e., what defines the route that goes from the initial mixture of DNA strands to the final intertwined superstructure, is, in some cases, still limited. In this review, we describe the main structural and energetic features of DNA nanoconstructs, from the simple Holliday junction to more complicated DNA architectures, and present the theoretical frameworks that have been formulated until now to explain their self-assembly. Deeper insights into the underlying principles of DNA self-assembly may certainly help us to overcome current experimental challenges and foster the development of original strategies inspired to dissipative and evolutive assembly processes occurring in nature.

2'-Fluoroarabinonucleic Acid Nanostructures as Stable Carriers for Cellular Delivery in the Strongly Acidic Environment.

DNA nanotechnology is powerful in constructing programmable nanostructures with distinct dimensions, sizes, and shapes. However, natural DNA molecules are prone to nuclease degradation, thus limiting the in vivo applications of such DNA nanostructures. 2'-Fluoroarabinonucleic acid (FANA) is a chemically modified oligonucleotide with similar base pairing properties to DNA and exhibits superior physical and chemical stabilities. In this work, FANA molecules were used to construct double crossover nanostructures, and it was demonstrated that incorporation of FANA conferred nucleic acid nanostructures with increased thermal stability and stronger nuclease resistance. More importantly, FANA nanostructures were able to maintain the structural integrity in the strongly acidic environment (pH 1.2). Last, such FANA nanostructures functioned well in acting as stable carriers of small-molecule cargoes for cellular delivery in simulated gastric fluid, while the DNA counterparts were mostly degraded. Collectively, these results demonstrated that FANA self-assembly was not only a substantial complement to the structural DNA nanotechnology but also an appealing molecular tool for in vivo biomedical applications.

Protein-Assisted Room-Temperature Assembly of Rigid, Immobile Holliday Junctions and Hierarchical DNA Nanostructures.

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redβ. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redβ to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redβ-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA–protein nanostructures may find applications in the crystallization of DNA–protein complexes, our work shows the great potential of Redβ to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.

DNA Self-Assembly Mediated by Programmable Soft-Patchy Interactions.

Adding shape and interaction anisotropy to a colloidal particle offers exquisitely tunable routes to engineer a rich assortment of complex-architected structures. Inspired by the hierarchical self-assembly concept with block copolymers and DNA liquid crystals and exploiting the unique assembly properties of DNA, we report here the construction and self-assembly of DNA-based soft-patchy anisotropic particles with a high degree of modularity in the system's design. By programmable positioning of thermoresponsive polymeric patches on the backbone of a stiff DNA duplex with linear and star-shaped architecture, we reversibly drive the DNA from a disordered ensemble to a diverse array of long-range ordered multidimensional nanostructures with tunable lattice spacing, ranging from lamellar to bicontinuous double-gyroid and double-diamond cubic morphologies, through the alteration of temperature. Our results demonstrate that the proposed hierarchical self-assembly strategy can be applied to any kind of DNA nanoarchitecture, highlighting the design principles for integration of self-assembly concepts from the physics of liquid crystals, block copolymers, and patchy colloids into the continuously growing interdisciplinary research field of structural DNA nanotechnology.

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.

Solvo-Driven Dimeric Nanoplasmon Coupling Under DNA Direction

Strong coupling is a prerequisite for nanoassemblies to deliver functions and applications unrealizable by noninteracting building units. DNA-Directed metamaterials, albeit highly programmable, oft...

Tetrahedral DNA nanostructures as drug delivery and bioimaging platforms in cancer therapy.

Structural DNA nanotechnology enables DNA to be used as nanomaterials for novel nanostructure construction with unprecedented functionalities. Artificial DNA nanostructures can be designed and generated with precisely controlled features, resulting in its utility in bionanotechnological and biomedical applications. A tetrahedral DNA nanostructure (TDN), the most popular DNA nanostructure, with high stability and simple synthesis procedure, is a promising candidate as nanocarriers in drug delivery and bioimaging platforms, particularly in precision medicine as well as diagnosis for cancer therapy. Recent evidence collectively indicated that TDN successfully enhanced cancer therapeutic efficiency both in vitro and in vivo. Here, we summarize the development of TDN and highlight various aspects of TDN applications in cancer therapy based on previous reports, including anticancer drug loading, photodynamic therapy, therapeutic oligonucleotides, bioimaging platforms, and other molecules and discuss a perspective in opportunities and challenges for future TDN‐based nanomedicine.

Modular self-assembly of gamma-modified peptide nucleic acids in organic solvent mixtures

Nucleic acid-based materials enable sub-nanometer precision in self-assembly for fields including biophysics, diagnostics, therapeutics, photonics, and nanofabrication. However, structural DNA nanotechnology has been limited to substantially hydrated media. Transfer to organic solvents commonly used in polymer and peptide synthesis results in the alteration of DNA helical structure or reduced thermal stabilities. Here we demonstrate that gamma-modified peptide nucleic acids (γPNA) can be used to enable formation of complex, self-assembling nanostructures in select polar aprotic organic solvent mixtures. However, unlike the diameter-monodisperse populations of nanofibers formed using analogous DNA approaches, γPNA structures appear to form bundles of nanofibers. A tight distribution of the nanofiber diameters could, however, be achieved in the presence of the surfactant SDS during self-assembly. We further demonstrate nanostructure morphology can be tuned by means of solvent solution and by strand substitution with DNA and unmodified PNA. This work thereby introduces a science of γPNA nanotechnology.

3D Freestanding DNA Nanostructure Hybrid as a Low-Density High-Strength Material.

Structural DNA nanotechnology can produce a wide range of 3D nanostructures with programmable structure and size at <5 nm resolution. However, it is challenging to dry these structures without capillary force-induced damage. As a result, the applications of 3D DNA nanostructures have long been limited in aqueous environments. Ready access to free-standing 3D DNA nanostructures in the dry state could revolutionize many research areas, especially in the development of low-density, high-strength materials. Here we report a method to obtain free-standing wireframe 3D DNA tetrahedra in air on a solid substrate, such as SiO2 and mica, by absorbing uranyl acetate and lyophilization. The dried DNA tetrahedron structure, 93 ± 2 nm in height, withstands 42 ± 22 nN of loading force. The effective hardness (9.1 ± 5.1 MPa) and Young's modulus (77 ± 48 MPa) of this low-density (70.7 kg/m3) DNA-inorganic hybrid nanostructure are comparable to other reported low-density high-strength materials.