DNA origami structures have great potential as functional platforms in various biomedical applications. Many applications, however, are incompatible with the high Mg concentrations commonly believed to be a prerequisite for maintaining DNA origami integrity. Herein, we investigate DNA origami stability in low-Mg buffers. DNA origami stability is found to crucially depend on the availability of residual Mg ions for screening electrostatic repulsion. The presence of EDTA and phosphate ions may thus facilitate DNA origami denaturation by displacing Mg ions from the DNA backbone and reducing the strength of the Mg -DNA interaction, respectively. Most remarkably, these buffer dependencies are affected by DNA origami superstructure. However, by rationally selecting buffer components and considering superstructure-dependent effects, the structural integrity of a given DNA origami nanostructure can be maintained in conventional buffers even at Mg concentrations in the low-micromolar range.

DNAo rigami structures have great potential as functional platforms in various biomedical applications.Many applications,h owever,a re incompatible with the high Mg 2+ concentrations commonly believed to be ap rerequisite for maintaining DNAo rigami integrity.H erein, we investigate DNAo rigami stability in low-Mg 2+ buffers.D NA origami stability is found to crucially depend on the availability of residual Mg 2+ ions for screening electrostatic repulsion. The presence of EDTAa nd phosphate ions may thus facilitate DNAo rigami denaturation by displacing Mg 2+ ions from the DNAb ackbone and reducing the strength of the Mg 2+ -DNA interaction, respectively.M ost remarkably,t hese buffer dependencies are affected by DNAo rigami superstructure. However,b yr ationally selecting buffer components and considering superstructure-dependent effects,t he structural integrity of ag iven DNAo rigami nanostructure can be maintained in conventional buffers even at Mg 2+ concentrations in the low-micromolar range.DNA origami [1,2] has become awidely used method for the fabrication of complex, yet well-defined nanostructures [3] with applications in biophysics, [4] molecular biology, [5] and drug and enzyme delivery. [6] Since many of these applications rely on intact DNAn anostructures,t he investigation of DNA origami stability under application-specific conditions has become am ajor research focus. [7][8][9][10][11] Biomedical applications in particular are often incompatible with the comparatively high (10-20 mm)M g 2+ concentrations required for DNA origami assembly.Onthe other hand, low-Mg 2+ concentration ( 1mm)h as been identified as one of the two most critical parameters that reduce DNAorigami stability in cell culture media. [8] Consequently,many approaches have been reported for protecting DNAo rigami nanostructures against destabilizing conditions and in particular low-Mg 2+ concentrations. [12][13][14] All the more surprising was the discovery that DNAorigami are stable in water for several weeks. [10] In these experiments,t he Mg 2+ -containing assembly buffer was exchanged for water through spin filtering,r esulting in Mg 2+ concentrations of afew micromolar.Although not completely Mg 2+ -free,m any applications may benefit from intact DNA origami nanostructures under such low-Mg 2+ conditions. However,other studies observed DNAorigami denaturation in buffers containing low-Mg 2+ concentrations. [7,8,11,12,14] These discrepancies could have various origins,s uch as differences in the buffer-exchange methods,buffer conditions, and DNAo rigami designs.I nt his work, we therefore investigate the stability of three DNAorigami nanostructures in as election of low-Mg 2+ buffers using the spin filteringbased buffer-exchange approach established by Linko et al. [10] We find that the composition of the buffer plays acritical role in DNAo rigami stability,w hile different DNAo rigami nanostructures show different buffer dependencies.First, we set out to reproduce the results of Linko et al. for the three DNAo rigami nanost...

The self-organized formation of regular patterns is not only a fascinating topic encountered in a multitude of natural and artificial systems, but also presents a versatile and powerful route toward large-scale nanostructure assembly and materials synthesis. The hierarchical, interface-assisted assembly of DNA origami nanostructures into regular, 2D lattices represents a particularly promising example, as the resulting lattices may exhibit an astonishing degree of order and can be further utilized as masks in molecular lithography. Here, we thus investigate the development of order in such 2D DNA origami lattices assembled on mica surfaces by employing in situ high-speed atomic force microscopy imaging. DNA origami lattice formation is found to resemble thin-film growth in several aspects. In particular, the Na+/Mg2+ ratio controls DNA origami adsorption, surface diffusion, and desorption, and is thus equivalent in its effects to substrate temperature which controls adatom dynamics in thin-film deposition. Consequently, we observe a pronounced dependence of lattice order on Na+ concentration. At low Na+ concentrations, lattice formation resembles random deposition and results in unordered monolayers, whereas very high Na+ concentrations are accompanied by rapid diffusion and especially DNA origami desorption, which prevent lattice formation. At intermediate Na+ concentrations, highly ordered DNA origami lattices are obtained that display an intricate symmetry, stemming from the complex shape of the employed Rothemund triangle. Nevertheless, even under such optimized conditions, the lattices display a considerable number of defects, including grain boundaries, point and line defects, and screw-like dislocations. By monitoring the dynamics of selected lattice defects, we identify mechanisms that limit the obtainable degree of lattice order. Possible routes toward further increasing lattice order by postassembly annealing are discussed.

Fibrinogen not only forms fibrin networks if assisted by thrombin but also exhibits self-assembly in dilute aqueous solutions in the absence of thrombin. It could be shown that self-assembly can be triggered in a controlled way by diluting the ionic strength set to a value of 0.14 M NaCl in the starting solutions. The present work unravels the mechanism of this self-assembly process by means of a combination of time-resolved multiangle static and dynamic light scattering and atomic force microscopy. Analysis was carried out as a function of the ionic strength adjusted by the drop in ionic strength and at variable salt compositions at a given final ionic strength. Composition was varied by changing the ratio of NaCl and phosphate buffer. The self-assembly induced by the drop of the ionic strength depends on the final value. The lower the final ionic strength gets, the faster is the self-assembly process. The variation of the salt composition at a given ionic strength has only a marginal effect, which depends on the ionic strength. The self-assembly obeys a step-growth process, where any intermediate cluster can coalesce with any other cluster. Interpretation of the data with a kinetic model based on the approach of von Smoluchowski follows a diffusion-limited cluster aggregation at ionic strength values lower than 30 mM. At an ionic strength of 30 mM, the model has to take into account a size dependence of the rate constant, and at 60 mM a transition is observed to a reaction-limited cluster aggregation.

Although DNA origami nanostructures have found their way into numerous fields of fundamental and applied research, they often suffer from rather limited stability when subjected to environments that differ from the employed assembly conditions, that is, suspended in Mg2+‐containing buffer at moderate temperatures. Here, means for efficient cryopreservation of 2D and 3D DNA origami nanostructures and, in particular, the effect of repeated freezing and thawing cycles are investigated. It is found that, while the 2D DNA origami nanostructures maintain their structural integrity over at least 32 freeze–thaw cycles, ice crystal formation makes the DNA origami gradually more sensitive toward harsh sample treatment conditions. Whereas no freeze damage could be detected in 3D DNA origami nanostructures subjected to 32 freeze–thaw cycles, 1000 freeze–thaw cycles result in significant fragmentation. The cryoprotectants glycerol and trehalose are found to efficiently protect the DNA origami nanostructures against freeze damage at concentrations between 0.2 × 10−3 and 200 × 10−3 m and without any negative effects on DNA origami shape. This work thus provides a basis for the long‐term storage of DNA origami nanostructures, which is an important prerequisite for various technological and medical applications.

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.

During the last decade, DNA origami nanostructures evolved into versatile platforms for the investigation of numerous biomolecular processes and reactions. [1] This development was initiated by the unique addressability of DNA origami nanostructures, which allows for the site-selective immobilization of multiple biomolecular species with nanometer resolution on the DNA origami surface. Using this approach, a vast variety of biomolecular processes have been investigated so far, including DNA-DNA [2] and DNA-RNA hybridization, [3] protein-DNA binding, [4] conformational transitions in DNA, [5] DNA radiation damage, [6] enzymatic DNA repair, [7] enzyme cascades, [8] antibody-antigen binding, [9-11] and even DNA transcription. [12] The aims of these studies were either to provide fundamental insights into the molecular mechanisms of biological processes or to improve the detection of medically relevant biomolecules. Another class of biomolecular reactions of great importance in a more technological context comprises the binding of proteins to small-molecule ligands, which is at the heart of drug screening and fragment-based drug discovery (FBDD). [13,14] The fundamental idea behind the FBDD approach is to identify and select few low-molecular mass fragments that efficiently bind to neighboring sites of the same target protein and subsequently merge them to construct a potent small-molecule protein inhibitor. [14] The latter step, however, often presents an enormous challenge as the linking chemistry

The rational combination of techniques from the fields of nanotechnology, single molecule detection, and lead discovery could provide elegant solutions to enhance the throughput of drug screening. We have synthesized nanoarrays of small pharmacophores on DNA origami substrates that are displayed either as individual ligands or as fragment pairs and thereby reduced the feature size by several orders of magnitude, as compared with standard microarray techniques. Atomic force microscopy-based single-molecule detection allowed us to distinguish potent protein-ligand interactions from weak binders. Several independent binding events, that is, strong, weak, symmetric bidentate, and asymmetric bidentate binding are directly visualized and evaluated. We apply this method to the discovery of bidentate trypsin binders based on benzamidine paired with aromatic fragments. Pairing of benzamidine with the dye TAMRA results in tenfold enhancement of the trypsin binding yield.