Effect of Staple Age on DNA Origami Nanostructure Assembly and Stability.
ABSTRACT: DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions. Furthermore, in the context of DNA origami mass production, the long-term storage of DNA origami nanostructures or their pre-assembled components also becomes an issue of high relevance, especially regarding the possible negative effects on DNA origami structural integrity. Thus, we investigated the effect of staple age on the self-assembly and stability of DNA origami nanostructures using atomic force microscopy. Different harsh processing conditions were simulated by applying different sample preparation protocols. Our results show that staple solutions may be stored at -20 °C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions. Mass spectrometry analysis of the aged staple mixtures revealed no signs of staple fragmentation. We, therefore, attribute the increased DNA origami sensitivity toward environmental conditions to an accumulation of damaged nucleobases, which undergo weaker base-pairing interactions and thus lead to reduced duplex stability.
Project description:Single-layer DNA origami is an efficient method for programmable self-assembly of nanostructures approximating almost any desired two-dimensional shape from ~5 MDa of DNA building material. In this method, a 7 kilobase single "scaffold" strand is assembled with hundreds of oligodeoxyribonucleotide "staple" strands to form a parallel array of double helices. Multiple layers of such DNA sheets also can be designed to assemble into a stack, enabling construction of solid three-dimensional shapes with considerably greater mechanical rigidity than two-dimensional shapes; however, the folding yield often is much lower and the required folding times are much longer. Here we introduce two strategies for designing multi-layer DNA origami that demonstrate potential for boosting assembly yield: (1) individual base pairs can be inserted between crossovers, allowing for greater bowing of helices at positions away from crossovers and therefore reduced electrostatic repulsion. At the same time, this underwinding of double helices increases a destabilizing torsional strain energy but then also increases affinity for intercalators, and binding of such intercalators can relieve this stress. We also have exploited this enhanced affinity for intercalators to PEGylate the surface of the nanostructures in a noncovalent fashion using PEG-tris-acridine. (2) Positioning of staple-strand breaks in the DNA origami such that each staple strand includes a 14 nucleotide (nt) continuous segment that binds to a complementary 14 nt continuous segment of the scaffold can greatly improve folding yields.
Project description:Scaffolded DNA origami enables the bottom-up fabrication of diverse DNA nanostructures by designing hundreds of staple strands, comprised of complementary sequences to the specific binding locations of a scaffold strand. Despite its exceptionally high design flexibility, poor reusability of staples has been one of the major hurdles to fabricate assorted DNA constructs in an effective way. Here we provide a rational module-based design approach to create distinct bent shapes with controllable geometries and flexibilities from a single, reference set of staples. By revising the staple connectivity within the desired module, we can control the location, stiffness, and included angle of hinges precisely, enabling the construction of dozens of single- or multiple-hinge structures with the replacement of staple strands up to 12.8% only. Our design approach, combined with computational shape prediction and analysis, can provide a versatile and cost-effective procedure in the design of DNA origami shapes with stiffness-tunable units.
Project description:Scaffolded DNA origami nanostructures enable the self-assembly of arbitrarily shaped objects with unprecedented accuracy. Yet, varying physiological conditions are prone to induce slight structural changes in the nanoscale architecture. Here, we report on high precision measurements of overall shape and interhelical distance of three prototypic DNA origami structures in solution using synchrotron small-angle X-ray scattering. Sheet-, brick-, and cylinder-shaped DNA constructs were assembled and the shape factors determined with angstrom resolution from fits to the scattering profiles. With decreasing MgCl2 concentration electrostatic swelling of both shape cross section and interhelical DNA spacing of the DNA origami structures is observed. The structures tolerate up to 10% interhelical expansion before they disintegrate. In contrast, with increasing temperature, the cylinder-shaped structures show no thermal expansion in a wide temperature window before they abruptly melt above 50 °C. Details on molecular structure of DNA origami can also be obtained using in-house X-ray scattering equipment and, hence, allow for routine folding and stability testing of DNA-based agents that are designed to operate under varying salt conditions.
Project description:Scaffolded DNA origami, a method to create self-assembled nanostructures with spatially addressable features, has recently been used to develop water-soluble molecular chips for label-free RNA detection, platforms for deterministic protein positioning, and single molecule reaction observatories. These applications highlight the possibility of exploiting the unique properties and biocompatibility of DNA nanostructures in live, cellular systems. Herein, we assembled several DNA origami nanostructures of differing shape, size and probes, and investigated their interaction with lysate obtained from various normal and cancerous cell lines. We separated and analyzed the origami-lysate mixtures using agarose gel electrophoresis and recovered the DNA structures for functional assay and subsequent microscopic examination. Our results demonstrate that DNA origami nanostructures are stable in cell lysate and can be easily separated from lysate mixtures, in contrast to natural, single- and double-stranded DNA. Atomic force microscope (AFM) and transmission electron microscope (TEM) images show that the DNA origami structures are fully intact after separation from cell lysates and hybridize to their targets, verifying the superior structural integrity and functionality of self-assembled DNA origami nanostructures relative to conventional oligonucleotides. The stability and functionality of DNA origami structures in cell lysate validate their use for biological applications, for example, as programmable molecular rafts or disease detection platforms.
Project description:Structural DNA nanotechnology utilizes DNA molecules as programmable information-coding polymers to create higher order structures at the nanometer scale. An important milestone in structural DNA nanotechnology was the development of scaffolded DNA origami in which a long single-stranded viral genome (scaffold strand) is folded into arbitrary shapes by hundreds of short synthetic oligonucleotides (staple strands). The achievable dimensions of the DNA origami tile units are currently limited by the length of the scaffold strand. Here we demonstrate a strategy referred to as "superorigami" or "origami of origami" to scale up DNA origami technology. First, this method uses a collection of bridge strands to prefold a single-stranded DNA scaffold into a loose framework. Subsequently, preformed individual DNA origami tiles are directed onto the loose framework so that each origami tile serves as a large staple. Using this strategy, we demonstrate the ability to organize DNA origami nanostructures into larger spatially addressable architectures.
Project description:DNA nanostructures, owing to their controllable and adaptable nature, have been considered as highly attractive nanoplatforms for biomedical applications in recent years. However, their use in the biological environment has been restricted by low cellular transfection efficiency in mammalian cells, weak stability under physiological conditions, and endonuclease degradation. Herein, we demonstrate an effective approach to facilitate fast transfection of DNA nanostructures and enhance their stability by encapsulating DNA origami with a biocompatible cationic protein (cHSA) via electrostatic interaction. The coated DNA origami is found to be stable under physiological conditions. Moreover, the cHSA coating could significantly improve the cellular transfection efficiency of DNA origami, which is essential for biological applications.
Project description:DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm(2). Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation.
Project description:Bottom-up fabrication of custom nanostructures using the methods of DNA nanotechnology has great potential for applications in many areas of science and technology. One obstacle to applications concerns the constrained environmental conditions at which DNA objects retain their structure. We present a general, site-selective, and scalable method for creating additional covalent bonds that increase the structural stability of DNA nanostructures. Placement of thymidines in close proximity within DNA nanostructures allows the rational creation of sites for covalent cyclobutane pyrimidine dimer (CPD) bonds induced via ultraviolet irradiation. The additional covalent bonds may be used in a sequence-programmable fashion to link free strand termini, to bridge strand breaks at crossover sites, and to create additional interhelical connections. Thus designed multilayer DNA origami objects can remain stable at temperatures up to 90°C and in pure double-distilled water with no additional cations present. In addition, these objects show enhanced resistance against nuclease activity. Cryo-electron microscopy (cryo-EM) structural analysis of non-cross-linked and cross-linked objects indicated that the global shape and the internal network of crossovers are preserved after irradiation. A cryo-EM map of a CPD-stabilized multilayer DNA origami object determined at physiological ionic strength reveals a substantial swelling behavior, presumably caused by repulsive electrostatic forces that, without covalent stabilization, would cause disassembly at low ionic strength. Our method opens new avenues for applications of DNA nanostructures in a wider range of conditions.
Project description:A versatile, bottom-up approach allows the controlled fabrication of polydopamine (PD) nanostructures on DNA origami. PD is a biosynthetic polymer that has been investigated as an adhesive and promising surface coating material. However, the control of dopamine polymerization is challenged by the multistage-mediated reaction mechanism and diverse chemical structures in PD. DNA origami decorated with multiple horseradish peroxidase-mimicking DNAzyme motifs was used to control the shape and size of PD formation with nanometer resolution. These fabricated PD nanostructures can serve as "supramolecular glue" for controlling DNA origami conformations. Facile liberation of the PD nanostructures from the DNA origami templates has been achieved in acidic medium. This presented DNA origami-controlled polymerization of a highly crosslinked polymer provides a unique access towards anisotropic PD architectures with distinct shapes that were retained even in the absence of the DNA origami template.
Project description:Investigations into the refolding of DNA origami leads to the creation of reconstructable nanostructures and deepens our understanding of the sustainability of life. Here, we report the refolding of the DNA origami structure inside a micron-sized compartment. In our experiments, conventional DNA origami and truss-type DNA origami were annealed and purified to remove the excess staples in a test tube. The DNA origami was then encapsulated inside of a micron-sized compartment of water-in-oil droplets, composed of neutral surfactants. The re-annealing process was then performed to initiate refolding in the compartment. The resulting 100-nm-sized DNA nanostructures were observed using atomic force microscopy (AFM), and the qualities of their structures were evaluated based on their shape. We found that the refolding of the DNA origami structure was favored inside the droplets compared with refolding in bulk solution. The refolded structures were able to fold even under "quick" one-minute annealing conditions. In addition, the smaller droplets (average diameter: 1.2 µm) appeared to be more advantageous for the refolding of the origamis than larger droplets. These results are expected to contribute to understanding the principles of life phenomena based on multimolecular polymer self-assembly in a micron-sized compartment, and for the production and maintenance of artificially designed molecules.