Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs.

Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.

Hepatitis B Virus Neutralization with DNA Origami Nanoshells.

We demonstrate the use of DNA origami to create virus-trapping nanoshells that efficiently neutralize hepatitis B virus (HBV) in cell culture. By modification of the shells with a synthetic monoclonal antibody that binds to the HBV envelope, the effective neutralization potency per antibody is increased by approximately 100 times compared to using free antibodies. The improvements in neutralizing the virus are attributed to two factors: first, the shells act as a physical barrier that blocks the virus from interacting with host cells; second, the multivalent binding of the antibodies inside the shells lead to stronger attachment to the trapped virus, a phenomenon known as avidity. Pre-incubation of shells with HBV and simultaneous addition of both components separately to cells lead to comparable levels of neutralization, indicating rapid trapping of the virions by the shells. Our study highlights the potential of the DNA shell system to rationally create antivirals using components that, when used individually, show little to no antiviral effectiveness.

Designing Rigid DNA Origami Templates for Molecular Visualization Using Cryo-EM.

DNA origami, a method for constructing nanostructures from DNA, offers potential for diverse scientific and technological applications due to its ability to integrate various molecular functionalities in a programmable manner. In this study, we examined the impact of internal crossover distribution and the compositional uniformity of staple strands on the structure of multilayer DNA origami using cryogenic electron microscopy (cryo-EM) single-particle analysis. A refined DNA object was utilized as an alignment framework in a host-guest model, where we successfully resolved an 8 kDa thrombin binding aptamer (TBA) linked to the host object. Our results broaden the spectrum of DNA in structural applications.

An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Hierarchical assembly is more robust than egalitarian assembly in synthetic capsids.

Self-assembly of complex and functional materials remains a grand challenge in soft material science. Efficient assembly depends on a delicate balance between thermodynamic and kinetic effects, requiring fine-tuning affinities and concentrations of subunits. By contrast, we introduce an assembly paradigm that allows large error-tolerance in the subunit affinity and helps avoid kinetic traps. Our combined experimental and computational approach uses a model system of triangular subunits programmed to assemble into T = 3 icosahedral capsids comprising 60 units. The experimental platform uses DNA origami to create monodisperse colloids whose three-dimensional geometry is controlled to nanometer precision, with two distinct bonds whose affinities are controlled to kBT precision, quantified in situ by static light scattering. The computational model uses a coarse-grained representation of subunits, short-ranged potentials, and Langevin dynamics. Experimental observations and modeling reveal that when the bond affinities are unequal, two distinct hierarchical assembly pathways occur, in which the subunits first form dimers in one case and pentamers in another. These hierarchical pathways produce complete capsids faster and are more robust against affinity variation than egalitarian pathways, in which all binding sites have equal strengths. This finding suggests that hierarchical assembly may be a general engineering principle for optimizing self-assembly of complex target structures.

A DNA turbine powered by a transmembrane potential across a nanopore.

Rotary motors play key roles in energy transduction, from macroscale windmills to nanoscale turbines such as ATP synthase in cells. Despite our abilities to construct engines at many scales, developing functional synthetic turbines at the nanoscale has remained challenging. Here, we experimentally demonstrate rationally designed nanoscale DNA origami turbines with three chiral blades. These DNA nanoturbines are 24-27 nm in height and diameter and can utilize transmembrane electrochemical potentials across nanopores to drive DNA bundles into sustained unidirectional rotations of up to 10 revolutions s-1. The rotation direction is set by the designed chirality of the turbine. All-atom molecular dynamics simulations show how hydrodynamic flows drive this turbine. At high salt concentrations, the rotation direction of turbines with the same chirality is reversed, which is explained by a change in the anisotropy of the electrophoretic mobility. Our artificial turbines operate autonomously in physiological conditions, converting energy from naturally abundant electrochemical potentials into mechanical work. The results open new possibilities for engineering active robotics at the nanoscale.

Programmable multispecific DNA-origami-based T-cell engagers.

Multispecific antibodies have emerged as versatile therapeutic agents, and therefore, approaches to optimize and streamline their design and assembly are needed. Here we report on the modular and programmable assembly of IgG antibodies, F(ab) and scFv fragments on DNA origami nanocarriers. We screened 105 distinct quadruplet antibody variants in vitro for the ability to activate T cells in the presence of target cells. T-cell engagers were identified, which in vitro showed the specific and efficient T-cell-mediated lysis of five distinct target cell lines. We used these T-cell engagers to target and lyse tumour cells in vivo in a xenograft mouse tumour model. Our approach enables the rapid generation, screening and testing of bi- and multispecific antibodies to facilitate preclinical pharmaceutical development from in vitro discovery to in vivo proof of concept.

A Laser-Driven Microrobot for Thermal Stimulation of Single Cells.

Here, the study presents a thermally activated cell-signal imaging (TACSI) microrobot, capable of photothermal actuation, sensing, and light-driven locomotion. The plasmonic soft microrobot is specifically designed for thermal stimulation of mammalian cells to investigate cell behavior under heat active conditions. Due to the integrated thermosensitive fluorescence probe, Rhodamine B, the system allows dynamic measurement of induced temperature changes. TACSI microrobots show excellent biocompatibility over 72 h in vitro, and they are capable of thermally activating single cells to cell clusters. Locomotion in a 3D workspace is achieved by relying on thermophoretic convection, and the microrobot speed is controlled within a range of 5-65 µm s-1 . In addition, light-driven actuation enables spatiotemporal control of the microrobot temperature up to a maximum of 60 °C. Using TACSI microrobots, this study targets single cells within a large population, and demonstrates thermal cell stimulation using calcium signaling as a biological output. Initial studies with human embryonic kidney 293 cells indicate a dose dependent change in intracellular calcium content within the photothermally controlled temperature range of 37-57 °C.

Triple-Stranded DNA As a Structural Element in DNA Origami.

Molecular self-assembly with DNA origami offers an attractive route to fabricate arbitrary three-dimensional nanostructures. In DNA origami, B-form double-helical DNA domains (dsDNA) are commonly linked with covalent phosphodiester strand crossovers to build up three-dimensional objects. To expand the palette of structural motifs in DNA origami, here we describe hybrid duplex-triplex DNA motifs as pH-dependent building blocks in DNA origami. We investigate design rules for incorporating triplex forming oligonucleotides and noncanonical duplex-triplex crossovers in multilayer DNA origami objects. We use single-particle cryoelectron microscopy to elucidate the structural basis of triplex domains and of duplex-triplex crossovers. We find that duplex-triplex crossovers can complement and fully replace the canonical duplex-duplex crossovers within DNA origami objects, for example, to increase the crossover density for potentially greater rigidity and reduced interhelical spacing, and to create connections at sites where conventional crossovers may be undesirable. We also show the pH-induced formation of a DNA origami object stabilized entirely by triplex-mediated strand crossovers.

Core-Shell Structured Fluorescent Protein Nanoparticles: New Paradigm Toward Zero-Thermal-Quenching in High-Power Biohybrid Light-Emitting Diodes.

Stable and efficient high-power biohybrid light-emitting diodes (Bio-HLEDs) using fluorescent proteins (FPs) in photon downconverting filters have not been achieved yet, reaching best efficiencies of 130 lm W-1 stable for >5 h. This is related to the rise of the device temperature (70-80 °C) caused by FP-motion and quick heat-transmission in water-based filters, they lead to a strong thermal emission quenching followed by the quick chromophore deactivation via photoinduced H-transfer. To tackle both issues at once, this work shows an elegant concept of a new FP-based nanoparticle, in which the FP core is shielded by a SiO2 -shell (FP@SiO2 ) with no loss of the photoluminescence figures-of-merit over years in foreign environments: dry powder at 25 °C (ambient) or constant 50 °C, as well as suspensions in organic solvents. This enables the preparation of water-free photon downconverting coatings with FP@SiO2 , realizing on-chip high-power Bio-HLEDs with 100 lm W-1 stable for >120 h. Both thermal emission quenching and H-transfer deactivation are suppressed, since the device temperature holds <40 °C and remote high-power Bio-HLEDs exhibit final stabilities of 130 days compared to reference devices with water-based FP@SiO2 (83 days) and FP-polymer coatings (>100 h). Hence, FP@SiO2 is a new paradigm toward water-free zero-thermal-quenching biophosphors for first-class high-power Bio-HLEDs.

Active Nuclear Import of Mammalian Cell-Expressible DNA Origami.

DNA origami enables the creation of complex 3D shapes from genetic material. Future uses could include the delivery of genetic instructions to cells, but nuclear import remains a major barrier to gene delivery due to the impermeability of the nuclear membrane. Here we realize active nuclear import of DNA origami objects in dividing and chemically arrested mammalian cells. We developed a custom DNA origami single-strand scaffold featuring a mammalian-cell expressible reporter gene (mCherry) and multiple Simian virus 40 (SV40) derived DNA nuclear targeting sequences (DTS). Inclusion of the DTS within DNA origami rescued gene expression in arrested cells, indicating that active transport into the nucleus occurs. Our work successfully adapts mechanisms known from viruses to promote the cellular expression of genetic instructions encoded within DNA origami objects.

Gene-encoding DNA origami for mammalian cell expression.

DNA origami may enable more versatile gene delivery applications through its ability to create custom nanoscale objects with specific targeting, cell-invading, and intracellular effector functionalities. Toward this goal here we describe the expression of genes folded in DNA origami objects delivered to mammalian cells. Genes readily express from custom-sequence single-strand scaffolds folded within DNA origami objects, provided that the objects can denature in the cell. We demonstrate enhanced gene expression efficiency by including and tuning multiple functional sequences and structures, including virus-inspired inverted-terminal repeat-like (ITR) hairpin motifs upstream or flanking the expression cassette. We describe gene-encoding DNA origami bricks that assemble into multimeric objects to enable stoichiometrically controlled co-delivery and expression of multiple genes in the same cells. Our work provides a framework for exploiting DNA origami for gene delivery applications.

Broad-Spectrum Virus Trapping with Heparan Sulfate-Modified DNA Origami Shells.

Effective broadband antiviral platforms that can act on existing viruses and viruses yet to emerge are not available, creating a need to explore treatment strategies beyond the trodden paths. Here, we report virus-encapsulating DNA origami shells that achieve broadband virus trapping properties by exploiting avidity and a widespread background affinity of viruses to heparan sulfate proteoglycans (HSPG). With a calibrated density of heparin and heparan sulfate (HS) derivatives crafted to the interior of DNA origami shells, we could encapsulate adeno, adeno-associated, chikungunya, dengue, human papilloma, noro, polio, rubella, and SARS-CoV-2 viruses or virus-like particles, in one and the same HS-functionalized shell system. Additional virus-type-specific binders were not needed for the trapping. Depending on the relative dimensions of shell to virus particles, multiple virus particles may be trapped per shell, and multiple shells can cover the surface of clusters of virus particles. The steric occlusion provided by the heparan sulfate-coated DNA origami shells can prevent viruses from further interactions with receptors, possibly including those found on cell surfaces.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.

Comparative analysis of extracellular vesicle isolation methods from human AML bone marrow cells and AML cell lines.

Cellular crosstalk between hematopoietic stem/progenitor cells and the bone marrow (BM) niche is vital for the development and maintenance of myeloid malignancies. These compartments can communicate via bidirectional transfer of extracellular vesicles (EVs). EV trafficking in acute myeloid leukemia (AML) plays a crucial role in shaping the BM microenvironment into a leukemia-permissive niche. Although several EV isolation methods have been developed, it remains a major challenge to define the most accurate and reliable procedure. Here, we tested the efficacy and functional assay compatibility of four different EV isolation methods in leukemia-derived EVs: (1) membrane affinity-based: exoEasy Kit alone and (2) in combination with Amicon filtration; (3) precipitation: ExoQuick-TC; and (4) ultracentrifugation (UC). Western blot analysis of EV fractions showed the highest enrichment of EV marker expression (e.g., CD63, HSP70, and TSG101) by precipitation with removal of overabundant soluble proteins [e.g., bovine serum albumin (BSA)], which were not discarded using UC. Besides the presence of damaged EVs after UC, intact EVs were successfully isolated with all methods as evidenced by highly maintained spherical- and cup-shaped vesicles in transmission electron microscopy. Nanoparticle tracking analysis of EV particle size and concentration revealed significant differences in EV isolation efficacy, with exoEasy Kit providing the highest EV yield recovery. Of note, functional assays with exoEasy Kit-isolated EVs showed significant toxicity towards treated target cells [e.g., mesenchymal stromal cells (MSCs)], which was abrogated when combining exoEasy Kit with Amicon filtration. Additionally, MSC treated with green fluorescent protein (GFP)-tagged exoEasy Kit-isolated EVs did not show any EV uptake, while EV isolation by precipitation demonstrated efficient EV internalization. Taken together, the choice of EV isolation procedure significantly impacts the yield and potential functionality of leukemia-derived EVs. The cheapest method (UC) resulted in contaminated and destructed EV fractions, while the isolation method with the highest EV yield (exoEasy Kit) appeared to be incompatible with functional assays. We identified two methods (precipitation-based ExoQuick-TC and membrane affinity-based exoEasy Kit combined with Amicon filtration) yielding pure and intact EVs, also suitable for application in functional assays. This study highlights the importance of selecting the right EV isolation method depending on the desired experimental design.

Geometrically programmed self-limited assembly of tubules using DNA origami colloids.

Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.

Phage-free production of artificial ssDNA with Escherichia coli.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L-1 of ssDNA in a stirred-tank bioreactor, thus realizing up to a 40-fold increase in maximal product concentration over previous scalable phage-free ssDNA production methods.

A DNA origami rotary ratchet motor.

To impart directionality to the motions of a molecular mechanism, one must overcome the random thermal forces that are ubiquitous on such small scales and in liquid solution at ambient temperature. In equilibrium without energy supply, directional motion cannot be sustained without violating the laws of thermodynamics. Under conditions away from thermodynamic equilibrium, directional motion may be achieved within the framework of Brownian ratchets, which are diffusive mechanisms that have broken inversion symmetry1-5. Ratcheting is thought to underpin the function of many natural biological motors, such as the F1F0-ATPase6-8, and it has been demonstrated experimentally in synthetic microscale systems (for example, to our knowledge, first in ref. 3) and also in artificial molecular motors created by organic chemical synthesis9-12. DNA nanotechnology13 has yielded a variety of nanoscale mechanisms, including pivots, hinges, crank sliders and rotary systems14-17, which can adopt different configurations, for example, triggered by strand-displacement reactions18,19 or by changing environmental parameters such as pH, ionic strength, temperature, external fields and by coupling their motions to those of natural motor proteins20-26. This previous work and considering low-Reynolds-number dynamics and inherent stochasticity27,28 led us to develop a nanoscale rotary motor built from DNA origami that is driven by ratcheting and whose mechanical capabilities approach those of biological motors such as F1F0-ATPase.

Nanoschalen aus DNA fangen und neutralisieren Viren.

The current pandemic has highlighted the need for new antiviral therapies, to respond to existing and emerging diseases transmitted by viral vectors. We developed a novel antiviral approach that is based on neutralizing viruses by trapping and encapsulation in artificial nano-shells. The surrounding shells prevent the interaction of viruses with host cells and thus interrupt an essential step in the lifecycle of most viruses.