Probing the role of sequence in the assembly of three‐dimensional DNA crystals

DNA is a widely used biopolymer for the construction of nanometer‐scale objects due to its programmability and structural predictability. One long‐standing goal of the DNA nanotechnology field has been the construction of three‐dimensional DNA crystals. We previously determined the X‐ray crystal structure of a DNA 13‐mer that forms a continuously hydrogen bonded three‐dimensional lattice through Watson‐Crick and non‐canonical base pairs. Our current study sets out to understand how the sequence of the Watson‐Crick duplex region influences crystallization of this 13‐mer. We screened all possible self‐complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson‐Crick pair influenced the crystallization propensity and the speed of crystal self‐assembly. We determined X‐ray crystal structures for 13 of these oligonucleotides and found sequence‐specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self‐assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self‐assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self‐assembly is nucleated by the formation of the Watson‐Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 618–626, 2015.     

Design, synthesis, and biocompatibility of an arginine-based polyester

Polycations are very useful in biotechnology. However, most existing polycations have high toxicity that significantly limits their clinical translation. We designed poly(ethylene argininylaspartate diglyceride) (PEAD) that is based on arginine, aspartic acid, glycerol, and ethylene glycol. A set of in vitro assays demonstrated that PEAD exhibited no cytotoxicity at 1 mg/mL, which is at least 100 times higher than the widely used polycation-polyethylenimine. Subcutaneous injection of 1 mg PEAD in rats did not cause an adverse response acutely or after 4 weeks. Zeta potential measurements revealed that PEAD has high affinity to biological polyanions such as DNA and hyaluronic acid. This polycation represents a new platform of biocompatible polycations that may lead to clinical innovations in gene therapy, controlled release, tissue engineering, biosensors, and medical devices.     

Are soil lichen communities structured by biotic interactions? A null model analysis

Question: Are soil lichen communities structured by biotic interactions? Location: Gypsum outcrops located next to Belmonte del Tajo, central Spain. Methods: We sampled a total of 68 (50 cm × 50 cm) plots in gypsum outcrops from central Spain. Each plot was divided into 100 (5 cm × 5 cm) sampling quadrats, and the presence of all lichen species in every quadrat was recorded (6800 quadrats in total). We used two realistic null models to generate random communities unstructured by biotic interactions, and used them to test the hypothesis that soil lichen species co‐occur less often than expected by chance. Results: We found fewer species combinations and less co‐occurrence than expected by chance. However, the latter result was dependent on the null model selected. The number of checkerboard pairs did not differ significantly from the null expectation. Conclusions: Overall, our results suggest that gypsiferous soil lichen communities are structured by competitive interactions. They are consistent with studies conducted with a wide variety of taxa, and fill a gap in our knowledge of the factors driving the small‐scale distribution of these important organisms.     

Spontaneous beta-barrel formation: an all-atom Monte Carlo study of Abeta16-22 oligomerization

Using all-atom Monte Carlo simulations with implicit water, combined with a cluster size analysis, we study the aggregation of Abeta(16) (-22), a peptide capable of forming amyloid fibrils. We consider a system of six initially randomly oriented Abeta(16) (-22) peptides, and investigate the thermodynamics and structural properties of aggregates formed by this system. The system is unaggregated without ordered secondary structure at high temperature, and forms beta-sheet rich aggregates at low temperature. At the crossover between these two regimes, we find that clusters of all sizes occur, whereas the beta-strand content is low. In one of several runs, we observe the spontaneous formation of a beta-barrel with six antiparallel strands. The beta-barrel stands out as the by far most long-lived aggregate seen in our simulations.     

Chromosome‐level hybrid de novo genome assemblies as an attainable option for nonmodel insects

The emergence of third‐generation sequencing (3GS; long‐reads) is bringing closer the goal of chromosome‐size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of nonmodel organisms. However, long‐read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short‐reads and long‐reads, provide an alternative efficient and cost‐effective approach to generate de novo, chromosome‐level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation are constantly being expanded and improved. This makes it difficult for nonexperts to find efficient, fast and tractable computational solutions for genome assembly, especially in the case of nonmodel organisms lacking a reference genome or one from a closely related species. In this study, we review and test the most recent pipelines for hybrid assemblies, comparing the model organism Drosophila melanogaster to a nonmodel cactophilic Drosophila, D. mojavensis. We show that it is possible to achieve excellent contiguity on this nonmodel organism using the dbg2olc pipeline.     

Theoretical aspects of virus capsid assembly

A virus capsid is constructed from many copies of the same protein(s). Molecular recognition is central to capsid assembly. The capsid protein must polymerize in order to create a three-dimensional protein polymer. More than structure is required to understand this self-assembly reaction: one must understand how the pieces come together in solution.     

De novo design of α,β-didehydrophenylalanine containing peptides: from models to applications

The de novo design of peptides and proteins has emerged as an approach for investigating protein structure and function. The success relies heavily on the ability to design relatively short peptides that can adopt stable secondary structures. To this end, substitution with α,β-dehydroamino acids, especially α,β-didehydrophenylalanine (ΔPhe or ΔF) has blossomed in manifold directions, providing a rich diversity of well-defined structural motifs. Introduction of α,β-didehydrophenylalanine induces β-bends in small and 3(10)-helices in longer peptide sequences. Most favorable conformation of ΔF residues are (φ,ψ) ～(60°, 30°), (-60°, -30°), (-60°, 150°), and (60°, -150°). These features have been exploited in designing helix-turn-helix, helical bundle arrangements, and glycine zipper type super secondary structural motifs. The unusual capability of α,β-didehydrophenylalanine ring to form a variety of multicentered interactions (N-H…O, C-H…O, C-H…π, and N-H…π) suggests its possible exploitation for future de novo design of supramolecular structures. This work has now been extended to the de novo design of peptides with antibiotic, antifibrillization activity, etc. More recently, self-assembling properties of small dehydropeptides have been explored. This review focuses primarily on the structural and functional behavior of α,β-didehydrophenylalanine containing peptides.     

Assembly of multimeric phage nanostructures through leucine zipper interactions

One barrier to the construction of nanoscale devices is the ability to place materials into 2D- and 3D-ordered arrays by controlling the assembly and ordering of connections between nanomaterials. Ordered assembly of nanoscale materials may potentially be achieved using biological tools that direct specific connections between individual components. Recently, viruses were successfully employed as scaffolds for the nucleation of nanoparticles and nanowires (Mao et al., 2004); however, there is a paucity of methods for the higher order assembly of phage-templated materials. Here we describe a general strategy for the assembly of filamentous bacteriophages into long, wire-like or into tripod-like structures. To prepare the linear phage assemblies, dimeric leucine zipper protein domains, fused to the p3 and p9 proteins of M13 bacteriophage, were employed to direct the specific end-to-end self-association of the bacteriophage particles. Electron microscopy revealed that up to 90% of the phage displaying complementary leucine zipper domains formed linear multi-phage assemblies, composed of up to 30 phage in length. To prepare tripod-like assemblies, phage were engineered to express trimeric leucine zippers as p3 fusion proteins. This resulted in 3D assembly with three individual phages attached at a single point. These ordered phage structures should provide a foundation for self-assembly of virally templated nanomaterials into useful devices.     

Thermally induced cyclization of L‐isoleucyl‐L‐alanine in solid state: Effect of dipeptide structure on reaction temperature and self‐assembly

Thermal treatment of short‐chain oligopeptides is able to initiate the process of their self‐assembly with the formation of organic nanostructures with unique properties. On the other hand, heating can lead to a chemical reaction with the formation of new substances with specific properties and ability to form structures with different morphology. Therefore, in order to have a desired process, researcher needs to find its temperature range. In the present work, cyclization of _L‐isoleucyl‐_L‐alanine dipeptide in the solid state upon heating was studied. Kinetic parameters of this reaction were estimated within the approaches of the nonisothermal kinetics. The correlation between side chain structure of dipeptides and temperature of their cyclization in the solid state was found for the first time. This correlation may be used to predict the temperature, at which dipeptide self‐assembly changes to chemical reaction. The differences in self‐assembly of linear and cyclic dipeptides were demonstrated using atomic force microscopy. The effect of dipeptide concentration in a source solution and an organic solvent used on self‐assembly of dipeptides was shown. The new information obtained on the thermal properties and self‐assembly of linear and cyclic forms of _L‐isoleucyl‐_L‐alanine may be useful for the design of new nanomaterials based on oligopeptides, as well as for the synthesis of cyclic oligopeptides.     

Two‐dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three‐dimensional crystals

Bacterial microcompartments (BMCs) are large intracellular bodies that serve as simple organelles in many bacteria. They are proteinaceous structures composed of key enzymes encapsulated by a polyhedral protein shell. In previous studies, the organization of these large shells has been inferred from the conserved packing of the component shell proteins in two‐dimensional (2D) layers within the context of three‐dimensional (3D) crystals. Here, we show that well‐ordered, 2D crystals of carboxysome shell proteins assemble spontaneously when His‐tagged proteins bind to a monolayer of nickelated lipid molecules at an air–water interface. The molecular packing within the 2D crystals recapitulates the layered hexagonal sheets observed in 3D crystals. The results reinforce current models for the molecular design of BMC shells.     

RADseq dataset with 90% missing data fully resolves recent radiation of Petalidium (Acanthaceae) in the ultra‐arid deserts of Namibia

Deserts, even those at tropical latitudes, often have strikingly low levels of plant diversity, particularly within genera. One remarkable exception to this pattern is the genus Petalidium (Acanthaceae), in which 37 of 40 named species occupy one of the driest environments on Earth, the Namib Desert of Namibia and neighboring Angola. To contribute to understanding this enigmatic diversity, we generated RADseq data for 47 accessions of Petalidium representing 22 species. We explored the impacts of 18 different combinations of assembly parameters in de novo assembly of the data across nine levels of missing data plus a best practice assembly using a reference Acanthaceae genome for a total of 171 sequence datasets assembled. RADseq data assembled at several thresholds of missing data, including 90% missing data, yielded phylogenetic hypotheses of Petalidium that were confidently and nearly fully resolved, which is notable given that divergence time analyses suggest a crown age for African species of 3.6–1.4 Ma. De novo assembly of our data yielded the most strongly supported and well‐resolved topologies; in contrast, reference‐based assembly performed poorly, perhaps due in part to moderate phylogenetic divergence between the reference genome, Ruellia speciosa, and the ingroup. Overall, we found that Petalidium, despite the harshness of the environment in which species occur, shows a net diversification rate (0.8–2.1 species per my) on par with those of diverse genera in tropical, Mediterranean, and alpine environments.     

Nanogels prepared by self-assembly of oppositely charged globular proteins

Ovalbumin and lysozyme are two main proteins in hen egg white with the isoelectric points of 4.8 and 11, respectively. Herein we report the manufacture of stable, narrowly distributed nanogels (hydrodynamic radius about 100 nm) using a novel and convenient method: ovalbumin and lysozyme solutions were mixed at pH 5.3, the mixture solution was adjusted to pH 10.3, then subsequently stirred and heated. The nanogels were characterized using a combination of techniques. The nanogels have spherical shape and core-shell structure. The core is mainly composed of lysozyme and the shell is mainly composed of ovalbumin. The proteins in the nanogels are in denatured states and they are bound by intermolecular hydrophobic interactions, hydrogen bonds, and disulfide bonds. The charges of the nanogels can be modulated by the pH of the medium. The electrostatic repulsion of ovalbumin molecules on the nanogel surface stabilizes the nanogels in aqueous solution. The formation mechanism of the nanogels is discussed.     

A study of the influence of polysaccharides on collagen self-assembly: nanostructure and kinetics

Collagen, a critical part of the extra-cellular matrix of tissues, is a popular native material for building scaffolding for tissue-engineering applications. To mimic the structural and functional profiles of materials found in the native extra-cellular matrix, numerous efforts have been made toward developing a novel scaffold combining collagen with other biomacromolecules. All of these works have been focused on improving the mechanical or biochemical properties of the collagen-based matrix. Unfortunately, most of these studies have failed to consider the nanostructure of collagen in the complex matrix. The aim of our study was to investigate the aggregation pattern of collagen after addition of polysaccharides with positive or negative charge, the dose-response relationship, and the effect on reconstitution kinetics. Generally, collagen self-assembles into fibrils with a diameter of around 95 nm but, in the presence of various polysaccharides in varying amounts, collagen self-assembles into different shapes with larger diameters compared with collagen alone. Although the morphology and diameter of the collagen fibrils varies with reconstitution conditions, the D-periods of the fibrils all remained the same regardless of the species or concentration of polysaccharides. The kinetics of fibril formation was determined from turbidity-time curves. All turbidity curves demonstrated that polysaccharides only alter the lag time and time frame of reconstitution, but have no significant effect on the mechanism of reconstitution. Together our data indicate that the presence of biomacromolecules can alter the kinetics and the 3D fibril ultrastructure of assembled collagen and that the consequent structural changes may affect cellular responses in medical applications.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A novel self‐assembling peptide with UV‐responsive properties

A novel heptapeptide comprising Ile‐Gln‐Ser‐Pro‐His‐Phe‐Phe (IQSPHFF) identified and found to undergo self‐assembly into microparticles in solution. To understand the effects of ultraviolet (UV) irradiation on the self‐assembly process, IQSPHFF solutions were exposed to the UV light of 365 nm at room temperature. This exposure was found to have a profound effect on the morphology of the self‐assembled aggregates, converting the microparticles to nanorod shapes. Circular dichroism and FTIR studies indicated distinct structural differences in the arrangements of the peptide moieties before and after UV irradiation. However, Mass spectrum analysis and high performance liquid chromatography of the peptide molecules before and after UV irradiation demonstrated that the chemical structure of IQSPHFF was not changed. UV–visible spectroscopy and fluorescence spectroscopy studies showed that the absorption peak both increased after UV irradiation. Overall, our data show that the heptapeptide with UV‐responsive properties. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 272–278, 2014.     

Identifying the causes and consequences of assembly gaps using a multiplatform genome assembly of a bird‐of‐paradise

Genome assemblies are currently being produced at an impressive rate by consortia and individual laboratories. The low costs and increasing efficiency of sequencing technologies now enable assembling genomes at unprecedented quality and contiguity. However, the difficulty in assembling repeat‐rich and GC‐rich regions (genomic “dark matter”) limits insights into the evolution of genome structure and regulatory networks. Here, we compare the efficiency of currently available sequencing technologies (short/linked/long reads and proximity ligation maps) and combinations thereof in assembling genomic dark matter. By adopting different de novo assembly strategies, we compare individual draft assemblies to a curated multiplatform reference assembly and identify the genomic features that cause gaps within each assembly. We show that a multiplatform assembly implementing long‐read, linked‐read and proximity sequencing technologies performs best at recovering transposable elements, multicopy MHC genes, GC‐rich microchromosomes and the repeat‐rich W chromosome. Telomere‐to‐telomere assemblies are not a reality yet for most organisms, but by leveraging technology choice it is now possible to minimize genome assembly gaps for downstream analysis. We provide a roadmap to tailor sequencing projects for optimized completeness of both the coding and noncoding parts of nonmodel genomes.     

Chromosomal‐level assembly of Takifugu obscurus (Abe, 1949) genome using third‐generation DNA sequencing and Hi‐C analysis

The Tetraodontidae family are known to have relatively small and compact genomes compared to other vertebrates. The obscure puffer fish Takifugu obscurus is an anadromous species that migrates to freshwater from the sea for spawning. Thus the euryhaline characteristics of T. obscurus have been investigated to gain understanding of their survival ability, osmoregulation, and other homeostatic mechanisms in both freshwater and seawater. In this study, a high quality chromosome‐level reference genome for T. obscurus was constructed using long‐read Pacific Biosciences (PacBio) Sequel sequencing and a Hi‐C‐based chromatin contact map platform. The final genome assembly of T. obscurus is 381 Mb, with a contig N50 length of 3,296 kb and longest length of 10.7 Mb, from a total of 62 Gb of raw reads generated using single‐molecule real‐time sequencing technology from a PacBio Sequel platform. The PacBio data were further clustered into chromosome‐scale scaffolds using a Hi‐C approach, resulting in a 373 Mb genome assembly with a contig N50 length of 15.2 Mb and and longest length of 28 Mb. When we directly compared the 22 longest scaffolds of T. obscurus to the 22 chromosomes of the tiger puffer Takifugu rubripes, a clear one‐to‐one orthologous relationship was observed between the two species, supporting the chromosome‐level assembly of T. obscurus. This genome assembly can serve as a valuable genetic resource for exploring fugu‐specific compact genome characteristics, and will provide essential genomic information for understanding molecular adaptations to salinity fluctuations and the evolution of osmoregulatory mechanisms.     

Self‐assembled nanostructures of long‐acting GnRH analogs modified at position 7

It is well known that GnRH analogs can self‐assemble into amyloid fibrils and that the duration of action of GnRH analogs depends on the ability of the amyloid to slowly release active peptides. The aim of this study was to investigate the influence of the amino acid residues at position 7 of GnRH analogues on peptide self‐assembly. It was found that the dominant shape of the nanostructure can be changed when the structures of the residues at position 7 differ significantly from that of leucine in Degarelix. When the backbone length was extended (peptide 9), or the side chain of the residue at position 7 was replaced by an aromatic ring (peptide 6), or the rotation of the amide bond was restricted (peptide 8), the nanostructure changed from fibrils to vesicles. The results also indicate that the increasing hydrophilicity had little influence on the nanostructure morphology. In addition, a suitable release rate was found to play a more important role for the duration of the peptide action by maintaining the equilibrium between the drug concentration and the persistent release time, while the nanostructure shape was found to exert little influence on the duration of the peptide action. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.