Cold denaturation of DNA origami nanostructures.

The coupling of structural transitions to heat capacity changes leads to destabilization of macromolecules at both elevated and lowered temperatures. DNA origami not only exhibit this property but also provide a nanoscopic observable of cold denaturation processes by directing intramolecular strain to the most sensitive elements within their hierarchical architecture.

Effect of DNA Origami Nanostructures on Bacterial Growth.

DNA origami nanostructures are a powerful tool in biomedicine and can be used to combat drug-resistant bacterial infections. However, the effect of unmodified DNA origami nanostructures on bacteria is yet to be elucidated. With the aim to obtain a better understanding of this phenomenon, the effect of three DNA origami shapes, i.e., DNA origami triangles, six-helix bundles (6HBs), and 24-helix bundles (24HBs), on the growth of Gram-negative Escherichia coli and Gram-positive Bacillus subtilis is investigated. The results reveal that while triangles and 24HBs can be used as a source of nutrients by E. coli and thereby promote population growth, their effect is much smaller than that of genomic single- and double-stranded DNA. However, no effect on E. coli population growth is observed for the 6HBs. On the other hand, B. subtilis does not show any significant changes in population growth when cultured with the different DNA origami shapes or genomic DNA. The detailed effect of DNA origami nanostructures on bacterial growth thus depends on the competence signals and uptake mechanism of each bacterial species, as well as the DNA origami shape. This should be considered in the development of antimicrobial DNA origami nanostructures.

Molecular Adhesion of a Pilus-Derived Peptide Involved in Pseudomonas aeruginosa Biofilm Formation on Non-Polar ZnO-Surfaces.

Bacterial colonization and biofilm formation on abiotic surfaces are initiated by the adhesion of peptides and proteins. Understanding the adhesion of such peptides and proteins at a molecular level thus represents an important step toward controlling and suppressing biofilm formation on technological and medical materials. This study investigates the molecular adhesion of a pilus-derived peptide that facilitates biofilm formation of Pseudomonas aeruginosa, a multidrug-resistant opportunistic pathogen frequently encountered in healthcare settings. Single-molecule force spectroscopy (SMFS) was performed on chemically etched ZnO 11 2 ‾ 0 ${\left(11\bar{2}0\right)}$ surfaces to gather insights about peptide adsorption force and its kinetics. Metal-free click chemistry for the fabrication of peptide-terminated SMFS cantilevers was performed on amine-terminated gold cantilevers and verified by X-ray photoelectron spectroscopy (XPS) and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). Atomic force microscopy (AFM) and XPS analyses reveal stable topographies and surface chemistries of the substrates that are not affected by SMFS. Rupture events described by the worm-like chain model (WLC) up to 600 pN were detected for the non-polar ZnO surfaces. The dissociation barrier energy at zero force ΔG(0), the transition state distance xb and bound-unbound dissociation rate at zero force koff (0) for the single crystalline substrate indicate that coordination and hydrogen bonds dominate the peptide/surface interaction.

Superstructure-dependent stability of DNA origami nanostructures in the presence of chaotropic denaturants.

The structural stability of DNA origami nanostructures in various chemical environments is an important factor in numerous applications, ranging from biomedicine and biophysics to analytical chemistry and materials synthesis. In this work, the stability of six different 2D and 3D DNA origami nanostructures is assessed in the presence of three different chaotropic salts, i.e., guanidinium sulfate (Gdm2SO4), guanidinium chloride (GdmCl), and tetrapropylammonium chloride (TPACl), which are widely employed denaturants. Using atomic force microscopy (AFM) to quantify nanostructural integrity, Gdm2SO4 is found to be the weakest and TPACl the strongest DNA origami denaturant, respectively. Despite different mechanisms of actions of the selected salts, DNA origami stability in each environment is observed to depend on DNA origami superstructure. This is especially pronounced for 3D DNA origami nanostructures, where mechanically more flexible designs show higher stability in both GdmCl and TPACl than more rigid ones. This is particularly remarkable as this general dependence has previously been observed under Mg2+-free conditions and may provide the possibility to optimize DNA origami design toward maximum stability in diverse chemical environments. Finally, it is demonstrated that melting temperature measurements may overestimate the stability of certain DNA origami nanostructures in certain chemical environments, so that such investigations should always be complemented by microscopic assessments of nanostructure integrity.

Adsorption of Ferritin at Nanofaceted Al2O3 Surfaces.

The influence of nanoscale surface topography on protein adsorption is highly important for numerous applications in medicine and technology. Herein, ferritin adsorption at flat and nanofaceted, single-crystalline Al2O3 surfaces is investigated using atomic force microscopy and X-ray photoelectron spectroscopy. The nanofaceted surfaces are generated by the thermal annealing of Al2O3 wafers at temperatures above 1000 °C, which leads to the formation of faceted saw-tooth-like surface topographies with periodicities of about 160 nm and amplitudes of about 15 nm. Ferritin adsorption at these nanofaceted surfaces is notably suppressed compared to the flat surface at a concentration of 10 mg/mL, which is attributed to lower adsorption affinities of the newly formed facets. Consequently, adsorption is restricted mostly to the pattern grooves, where the proteins can maximize their contact area with the surface. However, this effect depends on the protein concentration, with an inverse trend being observed at 30 mg/mL. Furthermore, different ferritin adsorption behavior is observed at topographically similar nanofacet patterns fabricated at different annealing temperatures and attributed to different step and kink densities. These results demonstrate that while protein adsorption at solid surfaces can be notably affected by nanofacet patterns, fine-tuning protein adsorption in this way requires the precise control of facet properties.

Multiprotein Adsorption from Human Serum at Gold and Oxidized Iron Surfaces Studied by Atomic Force Microscopy and Polarization-Modulation Infrared Reflection Absorption Spectroscopy.

Multiprotein adsorption from complex body fluids represents a highly important and complicated phenomenon in medicine. In this work, multiprotein adsorption from diluted human serum at gold and oxidized iron surfaces is investigated at different serum concentrations and pH values. Adsorption-induced changes in surface topography and the total amount of adsorbed proteins are quantified by atomic force microscopy (AFM) and polarization-modulation infrared reflection absorption spectroscopy (PM-IRRAS), respectively. For both surfaces, stronger protein adsorption is observed at pH 6 compared to pH 7 and pH 8. PM-IRRAS furthermore provides some qualitative insights into the pH-dependent alterations in the composition of the adsorbed multiprotein films. Changes in the amide II/amide I band area ratio and in particular side-chain IR absorption suggest that the increased adsorption at pH 6 is accompanied by a change in protein film composition. Presumably, this is mostly driven by the adsorption of human serum albumin, which at pH 6 adsorbs more readily and thereby replaces other proteins with lower surface affinities in the resulting multiprotein film.

Electrode Potential-Dependent Studies of Protein Adsorption on Ti6Al4V Alloy.

This article presents the potential-dependent adsorption of two proteins, bovine serum albumin (BSA) and lysozyme (LYZ), on Ti6Al4V alloy at pH 7.4 and 37 °C. The adsorption process was studied on an electropolished alloy under cathodic and anodic overpotentials, compared to the open circuit potential (OCP). To analyze the adsorption process, various complementary interface analytical techniques were employed, including PM-IRRAS (polarization-modulation infrared reflection-absorption spectroscopy), AFM (atomic force microscopy), XPS (X-ray photoelectron spectroscopy), and E-QCM (electrochemical quartz crystal microbalance) measurements. The polarization experiments were conducted within a potential range where charging of the electric double layer dominates, and Faradaic currents can be disregarded. The findings highlight the significant influence of the interfacial charge distribution on the adsorption of BSA and LYZ onto the alloy surface. Furthermore, electrochemical analysis of the protein layers formed under applied overpotentials demonstrated improved corrosion protection properties. These studies provide valuable insights into protein adsorption on titanium alloys under physiological conditions, characterized by varying potentials of the passive alloy.

Cation-dependent assembly of hexagonal DNA origami lattices on SiO2 surfaces.

DNA origami nanostructures have emerged as functional materials for applications in various areas of science and technology. In particular, the transfer of the DNA origami shape into inorganic materials using established silicon lithography methods holds great promise for the fabrication of nanostructured surfaces for nanoelectronics and nanophotonics. Using ordered DNA origami lattices directly assembled on the oxidized silicon surface instead of single nanostructures would enable the fabrication of functional nanopatterned surfaces with macroscopic dimensions. Here, we thus investigate the assembly of hexagonal DNA lattices from DNA origami triangles on RCA-cleaned silicon wafers with hydroxylated surface oxide by time-lapse atomic force microscopy (AFM). Lattice assembly on the SiO2 surface is achieved by a competition of monovalent and divalent cations at elevated temperatures. Ca2+ is found to be superior to Mg2+ in promoting the assembly of ordered lattices, while the presence of Mg2+ rather results in DNA origami aggregation and multilayer formation at the comparably high Na+ concentrations of 200 to 600 mM. Furthermore, Na+ concentration and temperature have a similar effect on lattice order, so that a reduction of temperature can be compensated to some extent by an increase in Na+ concentration. However, even under optimized conditions, the DNA origami lattices assembled on the SiO2 surface exhibit a lower degree of order than equivalent lattices assembled on mica, which is attributed to a higher desorption rate of the DNA origami nanostructures. Even though this high desorption rate also complicates any post-assembly treatment, the formed DNA origami lattices could successfully be transferred into the dry state, which is an important prerequisite for further processing steps.

Effect of Ionic Strength on the Thermal Stability of DNA Origami Nanostructures.

The stability of DNA origami nanostructures in aqueous media is closely tied to the presence of cations that screen electrostatic inter-helix repulsion. Here, the thermal melting behavior of different DNA origami nanostructures is investigated in dependence on Mg2+ concentration and compared to calculated ensemble melting temperatures of the staple strands used in DNA origami folding. Strong deviations of the measured DNA origami melting temperatures from the calculated ones are observed, in particular at high ionic strength where the melting temperature saturates and becomes independent of ionic strength. The degree of deviation between the measured and calculated melting temperatures further depends on the superstructure and in particular the mechanical properties of the DNA origami nanostructures. This indicates that thermal stability of a given DNA origami design at high ionic strength is governed predominantly not by electrostatic inter-helix repulsion but mostly by mechanical strain.

Stability of DNA Origami Nanostructures in Physiological Media: The Role of Molecular Interactions.

Programmable, custom-shaped, and nanometer-precise DNA origami nanostructures have rapidly emerged as prospective and versatile tools in bionanotechnology and biomedicine. Despite tremendous progress in their utilization in these fields, essential questions related to their structural stability under physiological conditions remain unanswered. Here, DNA origami stability is explored by strictly focusing on distinct molecular-level interactions. In this regard, the fundamental stabilizing and destabilizing ionic interactions as well as interactions involving various enzymes and other proteins are discussed, and their role in maintaining, modulating, or decreasing the structural integrity and colloidal stability of DNA origami nanostructures is summarized. Additionally, specific issues demanding further investigation are identified. This review - through its specific viewpoint - may serve as a primer for designing new, stable DNA objects and for adapting their use in applications dealing with physiological media.

Dynamics of DNA Origami Lattices.

Hierarchical assembly of programmable DNA frameworks─such as DNA origami─paves the way for versatile nanometer-precise parallel nanopatterning up to macroscopic scales. As of now, the rapid evolution of the DNA nanostructure design techniques and the accessibility of these methods provide a feasible platform for building highly ordered DNA-based assemblies for various purposes. So far, a plethora of different building blocks based on DNA tiles and DNA origami have been introduced, but the dynamics of the large-scale lattice assembly of such modules is still poorly understood. Here, we focus on the dynamics of two-dimensional surface-assisted DNA origami lattice assembly at mica and lipid substrates and the techniques for prospective three-dimensional assemblies, and finally, we summarize the potential applications of such systems.

Genotype-phenotype mapping with polyominos made from DNA origami tiles.

The correlation between genetic information and characteristics of a living cell-its genotype and its phenotype-constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a "1" and its absence to a "0." The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information ("genotype") and cluster morphology ("phenotype") thus reproduces key properties of genotype-phenotype maps known from living systems.

Time-Dependent DNA Origami Denaturation by Guanidinium Chloride, Guanidinium Sulfate, and Guanidinium Thiocyanate.

Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of DNA origami nanostructures by three Gdm salts, i.e., guanidinium chloride (GdmCl), guanidinium sulfate (Gdm2SO4), and guanidinium thiocyanate (GdmSCN), at different temperatures and in dependence of incubation time. Using DNA origami nanostructures as sensors that translate small molecular transitions into nanostructural changes, the denaturing effects of the Gdm salts were directly visualized by atomic force microscopy. GdmSCN was the most potent DNA denaturant, which caused complete DNA origami denaturation at 50 °C already at a concentration of 2 M. Under such harsh conditions, denaturation occurred within the first 15 min of Gdm exposure, whereas much slower kinetics were observed for the more weakly denaturing salt Gdm2SO4 at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in Gdm2SO4 with the fraction of intact nanostructures having an intermediate minimum at about 40 °C. Our results, thus, provide further insights into the highly complex Gdm-DNA interaction and underscore the importance of the counteranion species.

Effect of Surface Hydrophobicity on the Adsorption of a Pilus-Derived Adhesin-like Peptide.

Bacterial colonization of abiotic surfaces such as those of medical implants, membrane filters, and everyday household items is a process of tremendous importance for public health. Bacteria use adhesive cell surface structures called adhesins to establish contact with abiotic surfaces. Among them, protein filaments called type IV pili are particularly important and found in many Gram-negative pathogens such as Pseudomonas aeruginosa. Understanding the interaction of such adhesin proteins with different abiotic surfaces at the molecular level thus represents a fundamental prerequisite for impeding bacterial colonization and preventing the spread of infectious diseases. In this work, we investigate the interaction of a synthetic adhesin-like peptide, PAK128-144ox, derived from the type IV pilus of P. aeruginosa with hydrophilic and hydrophobic self-assembled monolayers (SAMs). Using a combination of molecular dynamics (MD) simulations, quartz crystal microbalance with dissipation monitoring (QCM-D), and spectroscopic investigations, we find that PAK128-144ox has a higher affinity for hydrophobic than for hydrophilic surfaces. Additionally, PAK128-144ox adsorption on the hydrophobic SAM is furthermore accompanied by a strong increase in α-helix content. Our results show a clear influence of surface hydrophobicity and further indicate that PAK128-144ox adsorption on the hydrophobic surface is enthalpically favored, while on the hydrophilic surface, entropic contributions are more significant. However, our spectroscopic investigations also suggest aggregation of the peptide under the employed experimental conditions, which is not considered in the MD simulations and should be addressed in more detail in future studies.

Direct visualization of the drug loading of single DNA origami nanostructures by AFM-IR nanospectroscopy.

The efficient loading of DNA nanostructures with intercalating or groove-binding drugs is an important prerequisite for various applications in drug delivery. However, unambiguous verification and quantification of successful drug loading is often rather challenging. In this work, AFM-IR nanospectroscopy is thus employed to directly visualize the loading of DNA origami nanostructures with the photosensitizer methylene blue (MB). Single MB-loaded DNA origami nanostructures can be clearly resolved in high-resolution infrared (IR) maps and the occurrence of MB-specific IR absorption correlates well with the topographic signals of the DNA origami nanostructures. The intensity of the recorded MB absorption bands furthermore scales with the MB concentration used for MB loading. By comparing single- and multilayer DNA origami nanostructures, it is also shown that the IR signal intensity of the loaded MB increases with the thickness of the DNA origami nanostructures. This indicates that also DNA double helices located in the core of bulky 3D DNA origami nanostructures are accessible for MB loading. AFM-IR nanospectroscopy thus has the potential to become an invaluable tool for quantifying drug loading of DNA origami nanostructures and optimizing drug loading protocols.

Anion-specific structure and stability of guanidinium-bound DNA origami.

While the folding of DNA into rationally designed DNA origami nanostructures has been studied extensively with the aim of increasing structural diversity and introducing functionality, the fundamental physical and chemical properties of these nanostructures remain largely elusive. Here, we investigate the correlation between atomistic, molecular, nanoscopic, and thermodynamic properties of DNA origami triangles. Using guanidinium (Gdm) as a DNA-stabilizing but potentially also denaturing cation, we explore the dependence of DNA origami stability on the identity of the accompanying anions. The statistical analyses of atomic force microscopy (AFM) images and circular dichroism (CD) spectra reveals that sulfate and chloride exert stabilizing and destabilizing effects, respectively, already below the global melting temperature of the DNA origami triangles. We identify structural transitions during thermal denaturation and show that heat capacity changes ΔC p determine the temperature sensitivity of structural damage. The different hydration shells of the anions and their potential to form Gdm+ ion pairs in concentrated salt solutions modulate ΔC p by altered wetting properties of hydrophobic DNA surface regions as shown by molecular dynamics simulations. The underlying structural changes on the molecular scale become amplified by the large number of structurally coupled DNA segments and thereby find nanoscopic correlations in AFM images.

Environment-Dependent Stability and Mechanical Properties of DNA Origami Six-Helix Bundles with Different Crossover Spacings.

The internal design of DNA nanostructures defines how they behave in different environmental conditions, such as endonuclease-rich or low-Mg2+ solutions. Notably, the inter-helical crossovers that form the core of such DNA objects have a major impact on their mechanical properties and stability. Importantly, crossover design can be used to optimize DNA nanostructures for target applications, especially when developing them for biomedical environments. To elucidate this, two otherwise identical DNA origami designs are presented that have a different number of staple crossovers between neighboring helices, spaced at 42- and 21- basepair (bp) intervals, respectively. The behavior of these structures is then compared in various buffer conditions, as well as when they are exposed to enzymatic digestion by DNase I. The results show that an increased number of crossovers significantly improves the nuclease resistance of the DNA origami by making it less accessible to digestion enzymes but simultaneously lowers its stability under Mg2+ -free conditions by reducing the malleability of the structures. Therefore, these results represent an important step toward rational, application-specific DNA nanostructure design.

The HSP40 chaperone Ydj1 drives amyloid beta 42 toxicity.

Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.

Salting-Out of DNA Origami Nanostructures by Ammonium Sulfate.

DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.

Magnesium-Free Immobilization of DNA Origami Nanostructures at Mica Surfaces for Atomic Force Microscopy.

DNA origami nanostructures (DONs) are promising substrates for the single-molecule investigation of biomolecular reactions and dynamics by in situ atomic force microscopy (AFM). For this, they are typically immobilized on mica substrates by adding millimolar concentrations of Mg2+ ions to the sample solution, which enable the adsorption of the negatively charged DONs at the like-charged mica surface. These non-physiological Mg2+ concentrations, however, present a serious limitation in such experiments as they may interfere with the reactions and processes under investigation. Therefore, we here evaluate three approaches to efficiently immobilize DONs at mica surfaces under essentially Mg2+-free conditions. These approaches rely on the pre-adsorption of different multivalent cations, i.e., Ni2+, poly-l-lysine (PLL), and spermidine (Spdn). DON adsorption is studied in phosphate-buffered saline (PBS) and pure water. In general, Ni2+ shows the worst performance with heavily deformed DONs. For 2D DON triangles, adsorption at PLL- and in particular Spdn-modified mica may outperform even Mg2+-mediated adsorption in terms of surface coverage, depending on the employed solution. For 3D six-helix bundles, less pronounced differences between the individual strategies are observed. Our results provide some general guidance for the immobilization of DONs at mica surfaces under Mg2+-free conditions and may aid future in situ AFM studies.