Engineered Protein Copolymers for Heparin Neutralization and Detection.

Heparin is a widely applied anticoagulant agent. However, in clinical practice, it is of vital importance to reverse its anticoagulant effect to restore the blood-clotting cascade and circumvent side effects. Inspired by protein cages that can encapsulate and protect their cargo from surroundings, we utilize three designed protein copolymers to sequester heparin into inert nanoparticles. In our design, a silk-like sequence provides cooperativity between proteins, generating a multivalency effect that enhances the heparin-binding ability. Protein copolymers complex heparin into well-defined nanoparticles with diameters below 200 nm. We also develop a competitive fluorescent switch-on assay for heparin detection, with a detection limit of 0.01 IU mL-1 in plasma that is significantly below the therapeutic range (0.2-8 IU mL-1). Moreover, moderate cytocompatibility is demonstrated by in vitro cell studies. Therefore, such engineered protein copolymers present a promising alternative for neutralizing and sensing heparin, but further optimization is required for in vivo applications.

Increasing Complexity in Wireframe DNA Nanostructures.

Structural DNA nanotechnology has recently gained significant momentum, as diverse design tools for producing custom DNA shapes have become more and more accessible to numerous laboratories worldwide. Most commonly, researchers are employing a scaffolded DNA origami technique by "sculpting" a desired shape from a given lattice composed of packed adjacent DNA helices. Albeit relatively straightforward to implement, this approach contains its own apparent restrictions. First, the designs are limited to certain lattice types. Second, the long scaffold strand that runs through the entire structure has to be manually routed. Third, the technique does not support trouble-free fabrication of hollow single-layer structures that may have more favorable features and properties compared to objects with closely packed helices, especially in biological research such as drug delivery. In this focused review, we discuss the recent development of wireframe DNA nanostructures-methods relying on meshing and rendering DNA-that may overcome these obstacles. In addition, we describe each available technique and the possible shapes that can be generated. Overall, the remarkable evolution in wireframe DNA structure design methods has not only induced an increase in their complexity and thus expanded the prevalent shape space, but also already reached a state at which the whole design process of a chosen shape can be carried out automatically. We believe that by combining cost-effective biotechnological mass production of DNA strands with top-down processes that decrease human input in the design procedure to minimum, this progress will lead us to a new era of DNA nanotechnology with potential applications coming increasingly into view.

High-Generation Amphiphilic Janus-Dendrimers as Stabilizing Agents for Drug Suspensions.

Pharmaceutical nanosuspensions are formed when drug crystals are suspended in aqueous media in the presence of stabilizers. This technology offers a convenient way to enhance the dissolution of poorly water-soluble drug compounds. The stabilizers exert their action through electrostatic or steric interactions, however, the molecular requirements of stabilizing agents have not been studied extensively. Here, four structurally related amphiphilic Janus-dendrimers were synthesized and screened to determine the roles of different macromolecular domains on the stabilization of drug crystals. Physical interaction and nanomilling experiments have substantiated that Janus-dendrimers with fourth generation hydrophilic dendrons were superior to third generation analogues and Poloxamer 188 in stabilizing indomethacin suspensions. Contact angle and surface plasmon resonance measurements support the hypothesis that Janus-dendrimers bind to indomethacin surfaces via hydrophobic interactions and that the number of hydrophobic alkyl tails determines the adsorption kinetics of the Janus-dendrimers. The results showed that amphiphilic Janus-dendrimers adsorb onto drug particles and thus can be used to provide steric stabilization against aggregation and recrystallization. The modular synthetic route for new amphiphilic Janus-dendrimers offers, thus, for the first time a versatile platform for stable general-use stabilizing agents of drug suspensions.

Dynamic DNA Origami Devices: from Strand-Displacement Reactions to External-Stimuli Responsive Systems.

DNA nanotechnology provides an excellent foundation for diverse nanoscale structures that can be used in various bioapplications and materials research. Among all existing DNA assembly techniques, DNA origami proves to be the most robust one for creating custom nanoshapes. Since its invention in 2006, building from the bottom up using DNA advanced drastically, and therefore, more and more complex DNA-based systems became accessible. So far, the vast majority of the demonstrated DNA origami frameworks are static by nature; however, there also exist dynamic DNA origami devices that are increasingly coming into view. In this review, we discuss DNA origami nanostructures that exhibit controlled translational or rotational movement when triggered by predefined DNA sequences, various molecular interactions, and/or external stimuli such as light, pH, temperature, and electromagnetic fields. The rapid evolution of such dynamic DNA origami tools will undoubtedly have a significant impact on molecular-scale precision measurements, targeted drug delivery and diagnostics; however, they can also play a role in the development of optical/plasmonic sensors, nanophotonic devices, and nanorobotics for numerous different tasks.

Self-assembly of amphiphilic Janus dendrimers into mechanically robust supramolecular hydrogels for sustained drug release.

Compounds that can gelate aqueous solutions offer an intriguing toolbox to create functional hydrogel materials for biomedical applications. Amphiphilic Janus dendrimers with low molecular weights can readily form self-assembled fibers at very low mass proportion (0.2 wt %) to create supramolecular hydrogels (G'≫G'') with outstanding mechanical properties and storage modulus of G'>1000 Pa. The G' value and gel melting temperature can be tuned by modulating the position or number of hydrophobic alkyl chains in the dendrimer structure; thus enabling exquisite control over the mesoscale material properties in these molecular assemblies. The gels are formed within seconds by simple injection of ethanol-solvated dendrimers into an aqueous solution. Cryogenic TEM, small-angle X-ray scattering, and SEM were used to confirm the fibrous structure morphology of the gels. Furthermore, the gels can be efficiently loaded with different bioactive cargo, such as active enzymes, peptides, or small-molecule drugs, to be used for sustained release in drug delivery.

3,4-Dimeth-oxy-4'-methyl-biphen-yl.

In the title compound, C15H16O2, the dihedral angle between the planes of the aromatic rings is 30.5 (2)°. In the crystal, mol-ecules are linked via C-H⋯O hydrogen bonds and C-H⋯π inter-actions, forming a two-dimensional network lying parallel to (100).

Methyl 3',4',5'-trimeth-oxy-biphenyl-4-carboxyl-ate.

In the title compound, C17H18O5, the dihedral angle between the benzene rings is 31.23 (16)°. In the crystal, the mol-ecules are packed in an anti-parallel fashion in layers along the a axis. In each layer, very weak C-H⋯O hydrogen bonds occur between the meth-oxy and methyl ester groups. Weak C-H⋯π inter-actions between the 4'- and 5'-meth-oxy groups and neighbouring benzene rings [meth-oxy-C-ring centroid distances = 4.075 and 3.486 Å, respectively] connect the layers.

Methyl 3',5'-dimeth-oxy-biphenyl-4-carboxyl-ate.

In the title compound, C16H16O4, the dihedral angle between the benzene rings is 28.9 (2)°. In the crystal, mol-ecules are packed in layers parallel to the b axis in which they are connected via weak inter-molecular C-H⋯O contacts. Face-to-face π-π inter-actions also exist between the benzene rings of adjacent mol-ecules, with centroid-centroid and plane-to-plane shift distances of 3.8597 (14) and 1.843 (2) Å, respectively.

3,5-Dimeth-oxy-4'-methyl-biphen-yl.

The title compound, C15H16O2, crystallizes with three independent mol-ecules in the asymmetric unit. The intra-molecular torsion angle between the aromatic rings of each mol-ecule are -36.4 (3), 41.3 (3) and -37.8 (3)°. In the crystal, the complicated packing of the mol-ecules forms wave-like layers along the b and c axes. The mol-ecules are connected via extensive meth-oxy-phenyl C-H⋯π inter-actions. A weak C-H⋯O hydrogen-bonding network also exists between meth-oxy O atoms and aromatic or meth-oxy H atoms.

3,4,5-Trimeth-oxy-4'-methyl-biphen-yl.

In the title compound, C16H18O3, the dihedral angle between the benzene rings is 33.4 (2)°. In the crystal, mol-ecules are packed in a zigzag arrangement along the b-axis and are inter-connected via weak C-H⋯O hydrogen bonds, and C-H⋯π inter-actions involving the meth-oxy groups and the benzene rings of neighbouring molecules.

Generic method for modular surface modification of cellulosic materials in aqueous medium by sequential "click" reaction and adsorption.

A generic approach for heterogeneous surface modification of cellulosic materials in aqueous medium, applicable for a wide range of functionalizations, is presented. In the first step, carboxymethyl cellulose (CMC) modified with azide or alkyne functionality, was adsorbed on a cellulosic substrate, thus, providing reactive sites for azide-alkyne cycloaddition click reactions. In the second step, functional units with complementary click units were reacted on the cellulose surface, coated by the click-modified CMC. Selected model functionalizations on diverse cellulosic substrates are shown to demonstrate the generality of the approach. The concept by sequentially combining the robust physical adsorption ("physical click") and robust chemical reaction ("chemical click") allows versatile, simple, and environmentally friendly modification of a cellulosic substrate with virtually any azide- or alkyne-modified molecule and even functionalization with several types of units.

Self-assembly of amphiphilic dendritic dipeptides into helical pores.

Natural pore-forming proteins act as viral helical coats and transmembrane channels, exhibit antibacterial activity and are used in synthetic systems, such as for reversible encapsulation or stochastic sensing. These diverse functions are intimately linked to protein structure. The close link between protein structure and protein function makes the design of synthetic mimics a formidable challenge, given that structure formation needs to be carefully controlled on all hierarchy levels, in solution and in the bulk. In fact, with few exceptions, synthetic pore structures capable of assembling into periodically ordered assemblies that are stable in solution and in the solid state have not yet been realized. In the case of dendrimers, covalent and non-covalent coating and assembly of a range of different structures has only yielded closed columns. Here we describe a library of amphiphilic dendritic dipeptides that self-assemble in solution and in bulk through a complex recognition process into helical pores. We find that the molecular recognition and self-assembly process is sufficiently robust to tolerate a range of modifications to the amphiphile structure, while preliminary proton transport measurements establish that the pores are functional. We expect that this class of self-assembling dendrimers will allow the design of a variety of biologically inspired systems with functional properties arising from their porous structure.

Advanced DNA Nanopore Technologies.

Diverse nanopore-based technologies have substantially expanded the toolbox for label-free single-molecule sensing and sequencing applications. Biological protein pores, lithographically fabricated solid-state and graphene nanopores, and hybrid pores are in widespread use and have proven to be feasible devices for detecting amino acids, polynucleotides, and their specific conformations. However, despite the indisputable and remarkable advantages in technological exploration and commercialization of such equipment, the commonly used methods may lack modularity and specificity in characterization of particular phenomena or in development of nanopore-based devices. In this review, we discuss DNA nanopore techniques that harness the extreme addressability, precision, and modularity of DNA nanostructures that can be incorporated as customized gates or plugs into for example lipid membranes, solid-state pores, and nanocapillaries, thus forming advanced hybrid instruments. In addition to these, there exist a number of diverse DNA-assisted nanopore-based detection and analysis methods. Here, we introduce different types of DNA nanostructure-based pore designs and their intriguing properties as well as summarize the extensive collection of current and future technologies and applications that can be realized through combining DNA nanotechnology with common nanopore approaches.

Robotic DNA Nanostructures.

Over the past decade, DNA nanotechnology has spawned a broad variety of functional nanostructures tailored toward the enabled state at which applications are coming increasingly in view. One of the branches of these applications is in synthetic biology, where the intrinsic programmability of the DNA nanostructures may pave the way for smart task-specific molecular robotics. In brief, the synthesis of the user-defined artificial DNA nano-objects is based on employing DNA molecules with custom lengths and sequences as building materials that predictably assemble together by obeying Watson-Crick base pairing rules. The general workflow of creating DNA nanoshapes is getting more and more straightforward, and some objects can be designed automatically from the top down. The versatile DNA nano-objects can serve as synthetic tools at the interface with biology, for example, in therapeutics and diagnostics as dynamic logic-gated nanopills, light-, pH-, and thermally driven devices. Such diverse apparatuses can also serve as optical polarizers, sensors and capsules, autonomous cargo-sorting robots, rotary machines, precision measurement tools, as well as electric and magnetic-field directed robotic arms. In this review, we summarize the recent progress in robotic DNA nanostructures, mechanics, and their various implementations.

Effect of double-tailed surfactant architecture on the conformation, self-assembly, and processing in polypeptide-surfactant complexes.

This work describes the solid-state conformational and structural properties of self-assembled polypeptide-surfactant complexes with double-tailed surfactants. Poly(L-lysine) was complexed with three dialkyl esters of phosphoric acid (i.e., phosphodiester surfactants), where the surfactant tail branching and length was varied to tune the supramolecular architecture in a facile way. After complexation with the branched surfactant bis(2-ethylhexyl) phosphate in an aqueous solution, the polypeptide chains adopted an alpha-helical conformation. These rod-like helices self-assembled into cylindrical phases with the amorphous alkyl tails pointing outward. In complexes with dioctyl phosphate and didodecyl phosphate, which have two linear n-octyl or n-dodecyl tails, respectively, the polypeptide formed antiparallel beta-sheets separated by alkyl layers, resulting in well-ordered lamellar self-assemblies. By heating, it was possible to trigger a partial opening of the beta-sheets and disruption of the lamellar phase. After repeated heating/cooling, all of these complexes also showed a glass transition between 37 and 50 degrees C. Organic solvent treatment and plasticization by overstoichiometric amount of surfactant led to structure modification in poly(L-lysine)-dioctyl phosphate complexes, PLL(diC8)(x) (x = 1.0-3.0). Here, the alpha-helical PLL is surrounded by the surfactants and these bottle-brush-like chains self-assemble in a hexagonal cylindrical morphology. As x is increased, the materials are clearly plasticized and the degree of ordering is improved: The stiff alpha-helical backbones in a softened surfactant matrix give rise to thermotropic liquid-crystalline phases. The complexes were examined by Fourier transform infrared spectroscopy, small- and wide-angle X-ray scattering, transmission electron microscopy, differential scanning calorimetry, polarized optical microscopy, and circular dichroism.

Hollow spherical supramolecular dendrimers.

The synthesis of a library containing 12 conical dendrons that self-assemble into hollow spherical supramolecular dendrimers is reported. The design principles for this library were accessed by development of a method that allows the identification of hollow spheres, followed by structural and retrostructural analysis of their Pm3n cubic lattice. The first hollow spherical supramolecular dendrimer was made by replacing the tapered dendron, from the previously reported tapered dendritic dipeptide that self-assembled into helical pores, with its constitutional isomeric conical dendron. This strategy generated a conical dendritic dipeptide that self-assembled into a hollow spherical supramolecular dendrimer that self-organizes in a Pm3n cubic lattice. Other examples of hollow spheres were assembled from conical dendrons without a dipeptide at their apex. These are conical dendrons originated from tapered dendrons containing additional benzyl ether groups at their apex. The inner part of the hollow sphere assembled from the dipeptide resembles the path of a spherical helix or loxodrome and, therefore, is chiral. The spheres assembled from other conical dendrons are nonhelical, even when they contain stereocenters on the alkyl groups from their periphery. Functionalization of the apex of the conical dendrons with diethylene glycol allowed the encapsulation of LiOTf and RbOTf in the center of the hollow sphere. These experiments showed that hollow spheres function as supramolecular dendritic capsules and therefore are expected to display functions complementary to those of other related molecular and supramolecular structures.

DNA nanostructure-directed assembly of metal nanoparticle superlattices.

Structural DNA nanotechnology provides unique, well-controlled, versatile, and highly addressable motifs and templates for assembling materials at the nanoscale. These methods to build from the bottom-up using DNA as a construction material are based on programmable and fully predictable Watson-Crick base pairing. Researchers have adopted these techniques to an increasing extent for creating numerous DNA nanostructures for a variety of uses ranging from nanoelectronics to drug-delivery applications. Recently, an increasing effort has been put into attaching nanoparticles (the size range of 1-20 nm) to the accurate DNA motifs and into creating metallic nanostructures (typically 20-100 nm) using designer DNA nanoshapes as molds or stencils. By combining nanoparticles with the superior addressability of DNA-based scaffolds, it is possible to form well-ordered materials with intriguing and completely new optical, plasmonic, electronic, and magnetic properties. This focused review discusses the DNA structure-directed nanoparticle assemblies covering the wide range of different one-, two-, and three-dimensional systems.

Evolution of Structural DNA Nanotechnology.

The research field entitled structural DNA nanotechnology emerged in the beginning of the 1980s as the first immobile synthetic nucleic acid junctions were postulated and demonstrated. Since then, the field has taken huge leaps toward advanced applications, especially during the past decade. This Progress Report summarizes how the controllable, custom, and accurate nanostructures have recently evolved together with powerful design and simulation software. Simultaneously they have provided a significant expansion of the shape space of the nanostructures. Today, researchers can select the most suitable fabrication methods, and design paradigms and software from a variety of options when creating unique DNA nanoobjects and shapes for a plethora of implementations in materials science, optics, plasmonics, molecular patterning, and nanomedicine.

Protein Coating of DNA Nanostructures for Enhanced Stability and Immunocompatibility.

Fully addressable DNA nanostructures, especially DNA origami, possess huge potential to serve as inherently biocompatible and versatile molecular platforms. However, their use as delivery vehicles in therapeutics is compromised by their low stability and poor transfection rates. This study shows that DNA origami can be coated by precisely defined one-to-one protein-dendron conjugates to tackle the aforementioned issues. The dendron part of the conjugate serves as a cationic binding domain that attaches to the negatively charged DNA origami surface via electrostatic interactions. The protein is attached to dendron through cysteine-maleimide bond, making the modular approach highly versatile. This work demonstrates the coating using two different proteins: bovine serum albumin (BSA) and class II hydrophobin (HFBI). The results reveal that BSA-coating significantly improves the origami stability against endonucleases (DNase I) and enhances the transfection into human embryonic kidney (HEK293) cells. Importantly, it is observed that BSA-coating attenuates the activation of immune response in mouse primary splenocytes. Serum albumin is the most abundant protein in the blood with a long circulation half-life and has already found clinically approved applications in drug delivery. It is therefore envisioned that the proposed system can open up further opportunities to tune the properties of DNA nanostructures in biological environment, and enable their use in various delivery applications.

Modular synthesis of self-assembling Janus-dendrimers and facile preparation of drug-loaded dendrimersomes.

Materials and methods aimed at the next generation of nanoscale carriers for drugs and other therapeutics are currently in great demand. Yet, creating these precise molecular arrangements in a feasible and straightforward manner represents a remarkable challenge. Herein we report a modular synthetic route for amphiphilic Janus-dendrimers via a copper-catalyzed click reaction (CuAAC) and a facile procedure, using simple injection, to obtain highly uniform dendrimersomes with efficient loading of the model drug compound propranolol. The resulting assemblies were analyzed by dynamic light scattering and cryogenic transmission electron microscopy revealing the formation of unilamellar and multilamellar dendrimersomes. The formation of a bilayer structure was confirmed using cryo-TEM and confocal microscopy visualization of an encapsulated solvatochromic dye (Nile Red). The dendrimersomes reported here are tunable in size, stable over time and display robust thermal stability in aqueous media. Our results expand the scope of dendrimer-based supramolecular colloidal systems and offer the means for one-step fabrication of drug-loaded dendrimersomes in the size range of 90-200 nm, ideal for biomedical applications.