A Critical View on the Use of DNA Hydrogels in Cell-Free Protein Synthesis.

Numerous studies have reported in the past that the use of protein-encoding DNA hydrogels as templates for cell-free protein synthesis (CFPS) leads to better yields than the use of conventional templates such as plasmids or PCR fragments. Systematic investigation of different types of bulk materials from pure DNA hydrogels and DNA hydrogel composites using a commercially available CFPS kit showed no evidence of improved expression efficiency. However, protein-coding DNA hydrogels were advantageously used in microfluidic reactors as immobilized templates for repetitive protein production, suggesting that DNA-based materials offer potential for future developments in high-throughput profiling or rapid in situ characterization of proteins.

Fluidic Interface for Surface-based DNA Origami Studies.

Traditionally, the use of DNA origami nanostructures (DONs) to study early cell signaling processes has been conducted using standard laboratory equipment with DONs typically utilized in solution. Surface-based technologies simplify the microscopic analysis of cells treated with DON agents by anchoring them to solid substrates, thus avoiding the complications of receptor-mediated endocytosis. A robust microfluidic platform for real-time monitoring and precise functionalization of surfaces with DONs was developed here.

Quantitative Characterization of RCA-Based DNA Hydrogels - Towards Rational Materials Design.

DNA hydrogels hold significant promise for biomedical applications and can be synthesized through enzymatic Rolling Circle Amplification (RCA). Due to the exploratory nature of this emerging field, standardized RCA protocols specifying the impact of reaction parameters are currently lacking. This study varied template sequences and reagent concentrations, evaluating RCA synthesis efficiency and hydrogel mechanical properties through quantitative PCR (qPCR) and indentation measurements, respectively.

Micromechanical Indentation Platform for Rapid Analysis of Viscoelastic Biomolecular Hydrogels.

The advent of biomedical applications of soft bioinspired materials has entailed an increasing demand for streamlined and expedient characterization methods meant for both research and quality control objectives. Here, a novel measurement system for the characterization of biological hydrogels with volumes as low as 75 µL was developed. The system is based on an indentation platform equipped with micrometer drive actuators that allow the determination of both the fracture points and Young's moduli of relatively stiff polymers, including agarose, as well as the measurements of viscosity for exceptionally soft and viscous hydrogels, such as DNA hydrogels.

Surface-Patterned DNA Origami Rulers Reveal Nanoscale Distance Dependency of the Epidermal Growth Factor Receptor Activation.

The nanoscale arrangement of ligands can have a major effect on the activation of membrane receptor proteins and thus cellular communication mechanisms. Here we report on the technological development and use of tailored DNA origami-based molecular rulers to fabricate "Multiscale Origami Structures As Interface for Cells" (MOSAIC), to enable the systematic investigation of the effect of the nanoscale spacing of epidermal growth factor (EGF) ligands on the activation of the EGF receptor (EGFR). MOSAIC-based analyses revealed that EGF distances of about 30-40 nm led to the highest response in EGFR activation of adherent MCF7 and Hela cells.

Nano- and Microscale Confinements in DNA-Scaffolded Enzyme Cascade Reactions.

Artificial reconstruction of naturally evolved principles, such as compartmentalization and cascading of multienzyme complexes, offers enormous potential for the development of biocatalytic materials and processes. Due to their unique addressability at the nanoscale, DNA origami nanostructures (DON) have proven to be an exceptionally powerful tool for studying the fundamental processes in biocatalytic cascades. To systematically investigate the diffusion-reaction network of (co)substrate transfer in enzyme cascades, a model system of stereoselective ketoreductase (KRED) with cofactor regenerating enzyme is assembled in different spatial arrangements on DNA nanostructures and is located in the sphere of microbeads (MB) as a spatially confining nano- and microenvironment, respectively.

Charge controlled interactions between DNA-modified silica nanoparticles and fluorosurfactants in microfluidic water-in-oil droplets.

Microfluidic droplets are an important tool for studying and mimicking biological systems, , to examine with high throughput the interaction of biomolecular components and the functionality of natural cells, or to develop basic principles for the engineering of artificial cells. Of particular importance is the approach to generate a biomimetic membrane by supramolecular self-assembly of nanoparticle components dissolved in the aqueous phase of the droplets at the inner water/oil interface, which can serve both to mechanically reinforce the droplets and as an interaction surface for cells and other components. While this interfacial assembly driven by electrostatic interaction of surfactants is quite well developed for water/mineral oil (W/MO) systems, no approaches have yet been described to exploit this principle for water/fluorocarbon oil (W/FO) emulsion droplets.

Activity-enhanced DNAzyme for design of label-free copper(II) biosensor.

Metal ion-driven, DNA-cleaving DNAzymes are characterised by high selectivity and specificity. However, their use for metal ion sensing remains largely unexplored due to long reaction times and poor reaction yields relative to RNA-cleaving DNAzymes and other sensing strategies. Herein we present a study demonstrating a significant rate enhancement of a copper-selective DNA cleaving DNAzyme by both polydopamine (PDA) and gold (Au) nanoparticles (NPs).

Selective Positioning of Different Cell Types on 3D Scaffolds via DNA Hybridization.

Three-dimensional (3D) microscaffolds for cell biology have shown their potential in mimicking physiological environments and simulating complex multicellular constructs. However, controlling the localization of cells precisely on microfabricated structures is still complex and usually limited to two-dimensional assays. Indeed, the implementation of an efficient method to selectively target different cell types to specific regions of a 3D microscaffold would represent a decisive step toward cell-by-cell assembly of complex cellular arrangements.

Orthogonal protein decoration of DNA nanostructures based on SpyCatcher-SpyTag interaction.

We present an efficient and readily applicable strategy for the covalent ligation of proteins to DNA origami by using the SpyCatcher-SpyTag (SC-ST) connector system. This approach showed orthogonality with other covalent connectors and has been used exemplarily for the immobilization and study of stereoselective ketoreductases to gain insight into the spatial arrangement of enzymes on DNA nanostructures.

Linker Engineering of Ligand-Decorated DNA Origami Nanostructures Affects Biological Activity.

News from an old acquaintance: The streptavidin (STV)-biotin binding system is frequently used for the decoration of DNA origami nanostructures (DON) to study biological systems. Here, a surprisingly high dynamic of the STV/DON interaction is reported, which is affected by the structure of the DNA linker system. Analysis of different mono- or bi-dentate linker architectures on DON with a novel high-speed atomic force microscope (HS-AFM) enabling acquisition times as short as 50 ms per frame gave detailed insights into the dynamics of the DON/STV interaction, revealing dwell times in the sub-100 millisecond range.

On the reproducibility of enzyme reactions and kinetic modelling.

Enzyme reactions are highly dependent on reaction conditions. To ensure reproducibility of enzyme reaction parameters, experiments need to be carefully designed and kinetic modeling meticulously executed. Furthermore, to enable quality control of enzyme reaction parameters, the experimental conditions, the modeling process as well as the raw data need to be reported comprehensively.

Nanowired electrodes as outer membrane cytochrome-independent electronic conduit in .

Extracellular electron transfer (EET) from microorganisms to inorganic electrodes is a unique ability of electrochemically active bacteria. Despite rigorous genetic and biochemical screening of the -type cytochromes that make up the EET network, the individual electron transfer steps over the cell membrane remain mostly unresolved. As such, attempts to transplant entire EET chains from native into non-native exoelectrogens have resulted in inferior electron transfer rates.

Enrichment of phosphate-accumulating organisms (PAOs) in a microfluidic model biofilm system by mimicking a typical aerobic granular sludge feast/famine regime.

Wastewater treatment using aerobic granular sludge has gained increasing interest due to its advantages compared to conventional activated sludge. The technology allows simultaneous removal of organic carbon, nitrogen, and phosphorus in a single reactor system and is independent of space-intensive settling tanks. However, due to the microscale, an analysis of processes and microbial population along the radius of granules is challenging.

DNA-Directed Assembly of a Cell-Responsive Biohybrid Interface for Cargo Release.

The development of a DNA-based cell-responsive biohybrid interface that can be used for spatially confined release of molecular cargo is reported. To this end, tailored DNA-protein conjugates are designed as gatekeepers that can be specifically cleaved by matrix metalloproteases (MMPs), which are secreted by many cancer cells. These gatekeepers can be installed by DNA hybridization on the surface of mesoporous silica nanoparticles (MSNs).

An Orthogonal Covalent Connector System for the Efficient Assembly of Enzyme Cascades on DNA Nanostructures.

Combining structural DNA nanotechnology with the virtually unlimited variety of enzymes offers unique opportunities for generating novel biocatalytic devices. However, the immobilization of enzymes is still restricted by a lack of efficient covalent coupling techniques. The rational re-engineering of the genetically fusible SNAP-tag linker is reported here.

Modeling-Assisted Design of Thermostable Benzaldehyde Lyases from Rhodococcus erythropolis for Continuous Production of α-Hydroxy Ketones.

Enantiopure α-hydroxy ketones are important building blocks of active pharmaceutical ingredients (APIs), which can be produced by thiamine-diphosphate-dependent lyases, such as benzaldehyde lyase. Here we report the discovery of a novel thermostable benzaldehyde lyase from Rhodococcus erythropolis R138 (ReBAL). While the overall sequence identity to the only experimentally confirmed benzaldehyde lyase from Pseudomonas fluorescens Biovar I (PfBAL) was only 65 %, comparison of a structural model of ReBAL with the crystal structure of PfBAL revealed only four divergent amino acids in the substrate binding cavity.

Formulation of DNA Nanocomposites: Towards Functional Materials for Protein Expression.

DNA hydrogels are an emerging class of materials that hold great promise for numerous biotechnological applications, ranging from tissue engineering to targeted drug delivery and cell-free protein synthesis (CFPS). In addition to the molecular programmability of DNA that can be used to instruct biological systems, the formulation of DNA materials, e.g.

Rapid quantitative assays for glucose-6-phosphate dehydrogenase (G6PD) and hemoglobin combined on a capillary-driven microfluidic chip.

Rapid tests for glucose-6-phosphate dehydrogenase (G6PD) are extremely important for determining G6PD deficiency, a widespread metabolic disorder which triggers hemolytic anemia in response to primaquine and tafenoquine medication, the most effective drugs for the radical cure of malaria caused by parasites. Current point-of-care diagnostic devices for G6PD are either qualitative, do not normalize G6PD activity to the hemoglobin concentration, or are very expensive. In this work we developed a capillary-driven microfluidic chip to perform a quantitative G6PD test and a hemoglobin measurement within 2 minutes and using less than 2 μL of sample.

Microfluidic cultivation and analysis of productive biofilms.

We here report the application of a machine-based microfluidic biofilm cultivation and analysis platform for studying the performance of biocatalytically active biofilms. By using robotic sampling, we succeeded in spatially resolving the productivity of three microfluidic reactors containing biocatalytically active biofilms that inducibly overexpress recombinant enzymes. Escherichia coli biofilms expressing two stereoselective oxidoreductases, the (R)-selective alcohol dehydrogenase LbADH and the (S)-selective ketoreductase Gre2p, as well as the phenolic acid decarboxylase EsPAD were used.

Meta-analysis of viscosity of aqueous deep eutectic solvents and their components.

Deep eutectic solvents (DES) formed by quaternary ammonium salts and hydrogen bond donors are a promising green alternative to organic solvents. Their high viscosity at ambient temperatures can limit biocatalytic applications and therefore requires fine-tuning by adjusting water content and temperature. Here, we performed a meta-analysis of the impact of water content and temperature on the viscosities of four deep eutectic solvents (glyceline, reline, N,N-diethylethanol ammonium chloride-glycerol, N,N-diethylethanol ammonium chloride-ethylene glycol), their components (choline chloride, urea, glycerol, ethylene glycol), methanol, and pure water.

Complex Nucleic Acid Hybridization Reactions inside Capillary-Driven Microfluidic Chips.

Nucleic acid hybridization reactions play an important role in many (bio)chemical fields, for example, for the development of portable point-of-care diagnostics, and often such applications require nucleic acid-based reaction systems that ideally run without enzymes under isothermal conditions. The use of novel capillary-driven microfluidic chips to perform two isothermal nucleic acid hybridization reactions, the simple opening of molecular beacon structures and the complex reaction cascade of a clamped-hybridization chain reaction (C-HCR), is reported here. For this purpose, reagents are arranged in a self-coalescence module (SCM) of a passive silicon microfluidic chip using inkjet spotting.

Postsynthetic Functionalization of DNA-Nanocomposites with Proteins Yields Bioinstructive Matrices for Cell Culture Applications.

We report on the directed postsynthetic functionalization of soft DNA nanocomposite materials with proteins. Using the example of the functionalization of silica nanoparticle-modified DNA polymer materials with agonists or antagonists of the epidermal growth factor receptor EGFR cell membrane receptor, we demonstrate that hierarchically structured interfaces to living cells can be established. Owing to the modular design principle, even complex DNA nanostructures can be integrated into the materials, thereby enabling the high-precision arrangement of ligands on the lower nanometer length scale.

Highly Modular Protein Micropatterning Sheds Light on the Role of Clathrin-Mediated Endocytosis for the Quantitative Analysis of Protein-Protein Interactions in Live Cells.

Protein micropatterning is a powerful tool for spatial arrangement of transmembrane and intracellular proteins in living cells. The restriction of one interaction partner (the bait, e.g.

Phosphoprotein Detection with a Single Nanofluidic Diode Decorated with Zinc Chelates.

We report a nanofluidic device for the label-free detection of phosphoprotein (PPn) analytes. To achieve this goal, a metal ion chelator, namely 4-[bis(2-pyridylmethyl)aminomethyl]aniline (DPA-NH ) compound was synthesized. Single asymmetric nanofluidic channels were fabricated in polyethylene terephthalate (PET) membranes.