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Actin is the most abundant protein in eukaryotic cells and is key to many cellular functions. The filamentous form of actin (F-actin) can be studied with help of natural products that specifically recognize it, as for example fluorophore-labeled probes of the bicyclic peptide phalloidin, but no synthetic probes exist for the monomeric form of actin (G-actin). Herein, we have panned a phage display library consisting of more than 10 billion bicyclic peptides against G-actin and isolated binders with low nanomolar affinity and greater than 1000-fold selectivity over F-actin. Sequence analysis revealed a strong similarity to a region of thymosin-β4, a protein that weakly binds G-actin, and competition binding experiments confirmed a common binding region at the cleft between actin subdomains 1 and 3. Together with F-actin-specific peptides that we also isolated, we evaluated the G-actin peptides as probes in pull-down, imaging, and competition binding experiments. While the F-actin peptides were applied successfully for capturing actin in cell lysates and for imaging, the G-actin peptides did not bind in the cellular context, most likely due to competition with thymosin-β4 or related endogenous proteins for the same binding site.

Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biohybrid hydrogels that change their mechanical properties in response to pharmacological cues hold high promises as externally controlled drug depots for biomedical applications. In this study, we devise a generically applicable method for the synthesis of micrometer-scale, injection-ready biohybrid materials. We use droplet-based microfluidics to generate monodisperse pre-microgel fluid droplets, wherein which we react fluorescein-modified 8-arm poly(ethylene glycol) with a thiol-functionalized humanized anti-fluorescein single chain antibody fragment and vinylsulfone-functionalized 8-arm poly(ethylene glycol), resulting in the formation of stable, narrowly dispersed supramolecular microgels (30 and 150 μm diameter). We demonstrate that the addition of free fluorescein to these microgels results in a weakening of their hydrogel structure, eventually leading to its disintegration. This method of formation of pharmacologically responsive biohybrid hydrogels in an injection-ready formulation is a pioneering example of a general approach for the synthesis of biohybrid hydrogel-based drug depots for biomedical applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Detecting drug-target interactions in real-time is a powerful approach for drug discovery and analytics. We show here for the first time the ultra fast electrical real-time detection and quantification of antibiotics using a novel biohybrid nanosensor. The biomolecular sensing is performed on ultralong (mm range) high aspect ratio nanowall (50 nm width) surfaces functionalized with operator DNA tetO which is specifically bound by the sensor protein TetR. This sensor protein is released from the operator DNA in a dose dependent manner by exposing the device functionalized with this bound DNA-protein complex to tetracycline antibiotics. As a result, the electrical conductance is accordingly modulated by these surface net charge changes. The switching mechanism of sensor proteins attached at the functionalized surfaces and releasing them again by antibiotics is demonstrated. With the here presented device the detection limit is below the limits of prevailing detection methods. Moreover, the study is extended to detect antibiotic residues in spiked organic milk from cows far below the maximum residual level of the European Union. In spiked milk samples a detection limit for tetracycline concentrations in the 100 fM level was achieved. The nanowall devices are fabricated by atomic layer deposition-based spacer lithography on full wafer scale which is a simple approach capable for mass production.

Biohybrid materials combining synthetic polymers with biological components are highly suited for tissue engineering in order to emulate the behavior of natural materials such as the extracellular matrix (ECM). In order to allow for an optimal cell-material interplay, the physical and biological parameters of the artificial matrix need to be dynamically remodeled during cultivation. Current tissue engineering concepts are mainly based on passive remodeling mechanisms including the degradation of the hydrogel and the release of incorporated biomolecules and therefore do not enable external adjustment of cultivation conditions. We present a novel hydrogel material that is able to serve as a cell growth matrix, whose degradation and presentation of cell-interacting biomolecules can be externally controlled by the addition of a pharmacological substance. The hydrogel is based on branched polyethylene glycol that is covalently decorated with the aminocoumarin-antibiotic switchable gyrase B protein conferring stimulus-responsive degradation. ECM properties were conferred to the hydrogels with cell attachment motifs and a general approach for the incorporation and inducible release of therapeutic biomolecules. This smart biohybrid material has the potential to serve as a next-generation tissue engineering device which allows for dynamic external adjustment of the physical and biological parameters, resulting in optimally controlled tissue formation., (Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Hydrogels provide a highly favorable matrix for immobilizing growth factors, enzymes or cells for biomedical applications like tissue engineering, drug delivery or the treatment of metabolic diseases. In this study we describe the synthesis and characterization of a hydrogel able to degrade L-ornithine, a metabolite that is highly elevated in congenital hyperornithinemia. The hydrogel was synthesized by embedding the L-ornithine-degrading enzymes L-ornithine aminotransferase (OAT) and L-ornithine decarboxylase (ODC) into a polymer network. The network was formed from linear polyacrylamide crosslinked by heterodimers of ODC and ornithine decarboxylase antizyme (OAz). The resulting hydrogel was shown to be stable under physiological conditions and to efficiently degrade L-ornithine. The hydrogel-stabilizing ODC-OAz interactions could subsequently be dissociated by the addition of antizyme inhibitor (AzI) which resulted in the inducible dissolution of the hydrogel. This L-ornithine-degrading hydrogel that can efficiently be eliminated when its functionality is no longer required might represent a first step towards an enzyme substitution approach against hyperornithinemia., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The synthetic reconstruction of natural gene networks and the de novo design of artificial genetic circuits provide new insights into the cell's regulatory mechanisms and will open new opportunities for drug discovery and intelligent therapeutic schemes. We will present how modular synthetic biology tools like repressors, promoters and enzymes can be assembled into complex systems in order to discover small molecules to shut off antibiotic resistance in tubercle bacteria and to design self-sufficient therapeutic networks. The transfer of these synthetic biological modules to the materials science field enables the construction of novel drug-inducible biohybrid materials for biomedical applications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The simplification of current vaccine administration regimes is of crucial interest in order to further sustain and expand the high impact of vaccines for public health. Most vaccines including the vaccine against hepatitis B need several doses to achieve protective immunization. In order to reduce the amount of repetitive injections, depot-based approaches represent a promising strategy. We present the application of novobiocin-sensitive biohybrid hydrogels as a depot for the pharmacologically controlled release of a vaccine against hepatitis B. Upon subcutaneous implantation of the vaccine depot into mice, we were able to release the vaccine by the oral administration of the stimulus molecule novobiocin resulting in successful immunization of the mice. This material-based vaccination regime holds high promises to replace classical vaccine injections conducted by medical personnel by the simple oral uptake of the stimulus thereby solving a major obstacle in increasing hepatitis B vaccination coverage.

The Cu(I)-catalyzed cycloaddition of terminal azides and alkynes (click chemistry) represents a highly specific reaction for the functionalization of biomolecules with chemical moieties such as dyes or polymer matrices. In this study we evaluate the use of bicinchoninic acid (BCA) as a ligand for Cu(I) under physiological reaction conditions. We demonstrate that the BCA-Cu(I)-complex represents an efficient catalyst for the conjugation of fluorophores or biotin to alkyne- or azide-functionalized proteins resulting in increased or at least equal reaction yields compared to commonly used catalysts like Cu(I) in complex with TBTA (tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine) or BPAA (bathophenanthroline disulfonic acid). The stabilization of Cu(I) with BCA represents a new strategy for achieving highly efficient bioconjugation reactions under physiological conditions in many application fields.

Interactive materials being responsive to a biocompatible stimulus represent a promising approach for future therapeutic applications. In this study, we present a novel biohybrid material synthesized from biocompatible components being stimulus-responsive to the pharmaceutically approved small-molecule novobiocin. The hydrogel design is based on the gyrase B (GyrB) protein, which is covalently grafted to multi-arm polyethylene glycol (PEG) using a Michael-type addition reaction. Upon addition of the GyrB-dimerizing substance coumermycin, stable hydrogels form which can be dissolved in a dose-adjustable manner by the antibiotic novobiocin. The switchable properties of this PEG-based hydrogel are favorable for future applications in tissue engineering and as externally controlled drug depot., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)