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.

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.

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.

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.

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.

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.

Self-Assembled Fibrinogen Hydro- and Aerogels with Fibrin-like 3D Structures.

The natural blood protein fibrinogen is a highly potent precursor for the production of various biomaterials due to its supreme biocompatibility and cell interaction. To gain actual materials from fibrinogen, the protein needs to undergo fibrillogenesis, which is mostly triggered via enzymatic processing to fibrin, electrospinning, or drying processes. All of those techniques, however, strongly limit the available structures or the applicability of the material. To overcome the current issues of fibrin(ogen) as material, we herein present a highly feasible, quick, and inexpensive technique for self-assembly of fibrinogen in solution into defined, nanofibrous three-dimensional (3D) patterns. Upon interaction with specific anions in controlled environments, stable and flexible hydrogel-like structures are formed without any further processing. Moreover, the material can be converted into highly porous and elastic aerogels by lyophilization. Both of these material classes have never been described before from native fibrinogen. The observed phenomenon also represents the first enzyme-free process of fibrillogenesis from fibrinogen with significant yield in solution. The produced hydrogels and aerogels were investigated via electron microscopy, IR spectroscopy, and fluorescence spectroscopy, which also confirms the native state of the protein. Additionally, their mechanical properties were compared with actual fibrin and unstructured fibrinogen. The structural features show a striking analogy to actual fibrin, both as hydro- and aerogel. This renders the new material a highly promising alternative for fibrin in biomaterial applications. A much faster initiation of fiber formation, exclusion of possible thrombin residuals, and low-cost reagents are great advantages.

Nanoscale Surface Topography Modulates hIAPP Aggregation Pathways at Solid-Liquid Interfaces.

The effects that solid-liquid interfaces exert on the aggregation of proteins and peptides are of high relevance for various fields of basic and applied research, ranging from molecular biology and biomedicine to nanotechnology. While the influence of surface chemistry has received a lot of attention in this context, the role of surface topography has mostly been neglected so far. In this work, therefore, we investigate the aggregation of the type 2 diabetes-associated peptide hormone hIAPP in contact with flat and nanopatterned silicon oxide surfaces. The nanopatterned surfaces are produced by ion beam irradiation, resulting in well-defined anisotropic ripple patterns with heights and periodicities of about 1.5 and 30 nm, respectively. Using time-lapse atomic force microscopy, the morphology of the hIAPP aggregates is characterized quantitatively. Aggregation results in both amorphous aggregates and amyloid fibrils, with the presence of the nanopatterns leading to retarded fibrillization and stronger amorphous aggregation. This is attributed to structural differences in the amorphous aggregates formed at the nanopatterned surface, which result in a lower propensity for nucleating amyloid fibrillization. Our results demonstrate that nanoscale surface topography may modulate peptide and protein aggregation pathways in complex and intricate ways.

Effect of DNA Origami Nanostructures on hIAPP Aggregation.

The aggregation of human islet amyloid polypeptide (hIAPP) plays a major role in the pathogenesis of type 2 diabetes mellitus (T2DM), and numerous strategies for controlling hIAPP aggregation have been investigated so far. In particular, several organic and inorganic nanoparticles (NPs) have shown the potential to influence the aggregation of hIAPP and other amyloidogenic proteins and peptides. In addition to conventional NPs, DNA nanostructures are receiving more and more attention from the biomedical field. Therefore, in this work, we investigated the effects of two different DNA origami nanostructures on hIAPP aggregation. To this end, we employed in situ turbidity measurements and ex situ atomic force microscopy (AFM). The turbidity measurements revealed a retarding effect of the DNA nanostructures on hIAPP aggregation, while the AFM results showed the co-aggregation of hIAPP with the DNA origami nanostructures into hybrid peptide-DNA aggregates. We assume that this was caused by strong electrostatic interactions between the negatively charged DNA origami nanostructures and the positively charged peptide. Most intriguingly, the influence of the DNA origami nanostructures on hIAPP aggregation differed from that of genomic double-stranded DNA (dsDNA) and appeared to depend on DNA origami superstructure. DNA origami nanostructures may thus represent a novel route for modulating amyloid aggregation in vivo.

Effect of Terminal Modifications on the Adsorption and Assembly of hIAPP(20-29).

The assembly of peptides and proteins into nanoscale amyloid fibrils via formation of intermolecular ß-sheets not only plays an important role in the development of degenerative diseases but also represents a promising approach for the synthesis of functional nanomaterials. In many biological and technological settings, peptide assembly occurs in the presence of organic and inorganic interfaces with different physicochemical properties. In an attempt to dissect the relative contributions of the different molecular interactions governing amyloid assembly at interfaces, we here present a systematic study of the effects of terminal modifications on the adsorption and assembly of the human islet amyloid polypeptide fragment hIAPP(20-29) at organic self-assembled monolayers (SAMs) presenting different functional groups (cationic, anionic, polar, or hydrophobic). Using a selection of complementary in situ and ex situ analytical techniques, we find that even this well-defined and comparatively simple model system is governed by a rather complex interplay of electrostatic interactions, hydrophobic interactions, and hydrogen bonding, resulting in a plethora of observations and dependencies, some of which are rather counterintuitive. In particular, our results demonstrate that terminal modifications can have tremendous effects on peptide adsorption and assembly dynamics, as well as aggregate morphology and molecular structure. The effects exerted by the terminal modifications can furthermore be modulated in nontrivial ways by the physicochemical properties of the SAM surface. Therefore, terminal modifications are an important factor to consider when conducting and comparing peptide adsorption and aggregation studies and may represent an additional parameter for guiding the assembly of peptide-based nanomaterials.

On the Adsorption of DNA Origami Nanostructures in Nanohole Arrays.

DNA origami nanostructures are versatile substrates for the controlled arrangement of molecular capture sites with nanometer precision and thus have many promising applications in single-molecule bioanalysis. Here, we investigate the adsorption of DNA origami nanostructures in nanohole arrays which represent an important class of biosensors and may benefit from the incorporation of DNA origami-based molecular probes. Nanoholes with well-defined diameter that enable the adsorption of single DNA origami triangles are fabricated in Au films on Si wafers by nanosphere lithography. The efficiency of directed DNA origami adsorption on the exposed SiO2 areas at the bottoms of the nanoholes is evaluated in dependence of various parameters, i.e., Mg2+ and DNA origami concentrations, buffer strength, adsorption time, and nanohole diameter. We observe that the buffer strength has a surprisingly strong effect on DNA origami adsorption in the nanoholes and that multiple DNA origami triangles with 120 nm edge length can adsorb in nanoholes as small as 120 nm in diameter. We attribute the latter observation to the low lateral mobility of once adsorbed DNA origami on the SiO2 surface, in combination with parasitic adsorption to the Au film. Although parasitic adsorption can be suppressed by modifying the Au film with a hydrophobic self-assembled monolayer, the limited surface mobility of the adsorbed DNA origami still leads to poor localization accuracy in the nanoholes and results in many DNA origami crossing the boundary to the Au film even under optimized conditions. We discuss possible ways to minimize this effect by varying the composition of the adsorption buffer, employing different fabrication conditions, or using other substrate materials for nanohole array fabrication.

Adsorption and Fibrillization of Islet Amyloid Polypeptide at Self-Assembled Monolayers Studied by QCM-D, AFM, and PM-IRRAS.

Aggregation and fibrillization of human islet amyloid polypeptide (hIAPP) plays an important role in the development of type 2 diabetes mellitus. Understanding the interaction of hIAPP with interfaces such as cell membranes at a molecular level therefore represents an important step toward new therapies. Here, we investigate the fibrillization of hIAPP at different self-assembled alkanethiol monolayers (SAMs) by quartz crystal microbalance with dissipation monitoring (QCM-D), atomic force microscopy (AFM), and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). We find that hydrophobic interactions with the CH3-terminated SAM tend to retard hIAPP fibrillization compared to the carboxylic acid-terminated SAM where attractive electrostatic interactions lead to the formation of a three-dimensional network of interwoven fibrils. At the hydroxyl- and amino-terminated SAMs, fibrillization appears to be governed by hydrogen bonding between the peptide and the terminating groups which may even overcome electrostatic repulsion. These results thus provide fundamental insights into the molecular mechanisms governing amyloid assembly at interfaces.