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| Beschreibung | [methodology] |
|---|---|
Artificial Protein Scaffolds for Controlled Assembly of Supramolecular Complexes. |
Artificial protein complexes are of high interest for the build-up of molecular factories because they enable assembly of multiple functional subunits in close proximity to one another, providing enhanced catalysis of chemical reactions in series. Assembly of binding proteins (e.g., antibodies) onto scaffolds is also highly desirable for delivery of protein therapeutics because the multi-valent complexes exhibit increased bound lifetime, effectively increasing the apparent affinity of a specific binding molecule to the cell surface. A major limitation, however, remains the preparation of large scaffolds onto which enzymes and antibodies can assemble. Several strategies have been developed for synthesizing scaffolds, for example, using repeated copies of receptor proteins, or DNA-based scaffolds. These approaches, however, are limited in terms of scaffold size, specificity and/or stability. The Nash Group synthesizes molecular scaffolds using repetitive protein building blocks called elastin-like polypeptides, and incorporates bioorthogonal functional groups for linkage to proteins and enzymes. The outcomes will be scaffold proteins which provide a versatile platform that is generalizable to many different reaction cascades and binding molecules. This work represents a simplified and scalable process to generate supramolecular complexes with novel functionality. |
Constraining chemical reactions into physical compartments enables spatial control of reactants, stringent variation of reaction conditions and possibilities to locally apply electrical, electrochemical or optical stimuli. Such external “smart reagents” can reach high intensities and high gradients at nanoscale dimensions and allow thereby production rates to be regulated, reactivities to be switched on and off, selective reaction pathways to be chosen or even novel synthetic mechanisms to be introduced. |
By top-down nanofabrication of scalable semiconductor devices with fluidic, electrical, electrochemical and optical access and site-selective functionalization by bottom-up molecular engineering, we can create molecular factories being able to produce added-value chemicals. Our solid-state compartmentalization approach leverages the high solvent compatibility and seamless sensing- and trigger-integration capability of silicon semiconductor platforms that we design, manufacture and package in-house in IBM’s Binnig and Rohrer Nanotechnology Center and IBM’s Noise-free Labs. Such silicon-based devices are used for a variety of tasks - ranging from fundamental science to applied research - in joint efforts within the NCCR MSE network: Interlinked Reaction Compartments with Molecular Functionalitiesjoint research activity with Mayor group
Site-selective Functionalization of Metal and Si-surfacesjoint research activity with Mayor group
Optical Sensing of Molecular Binding Interactions joint research activity with Mayor group
External Oriented Electric Field-assisted Chemistryjoint research activity with Mayor group
Divergent Compound Library Synthesis by Cascaded Heterogeneous Catalysisjoint research activity with Sparr and Mayor groups
Apart from these activities, tailored silicon-based microfluidic hybrid devices, e.g. Silicon-on-insulator/glass devices with optical viewports and electrodes, are produced for oil-water and polymer-membrane soft-compartment generation, pico-injection and real-time sorting in a collaboration with the Panke, Pallivan and Meyer groups. Additional funding is used in a close collaboration with the Fussenegger group on the development of an electrogenetic implant device that enables feedback control and telemetrical linkage to cognitive systems. See more details here. |
Our lab has pioneered the use of direct perfusion of fluid through the matrix of engineered tissues to mimic interstitial fluid flow within custom-designed bioreactor chambers in order to generate and sustain uniform tissue structures. |
Diabetes mellitus, the complex and multifactorial disease characterized by hyperglycaemia, results from a loss of pancreatic insulin-producing β-cells. However, none of the current cell-based diabetic therapies is autologous; whereas transplantation of pancreatic islets typically suffers from donor scarcity, compatibility and variability in graft quality. A way to solve these issues is to engineer autologous patient cells to act like healthy pancreatic β-cells and re-implant them. We hypothesize that culture of these genetically programmed pancreatic cells within the developed 3D perfusion-based culture system in co-culture with vascularizing autologous cells will generate micro-tissues with improved in vitro and in vivo functionality. |
We are creating and exploring nano-reaction chambers as catalysts for substrate and product selective reactions. Such functional modules will be the basis for the construction of molecular factories. |
The successful creation of a molecular factory relies on the delicate interplay of all its subcomponents. To avoid undesired cross-reactivity, the subcomponents have to display high substrate selectivity. One way to achieve substrate selectivity is the utilization of nano-reaction chambers, which due to their limited cavity volume only convert molecules of suitable size. Beside substrate selectivity, nano-reaction chambers also provide product selectivity. Due to the restricted environment inside the catalyst, the substrate conformation is influenced, which can lead to different products than in a regular solution experiment. Additionally, weak interactions between the host and the guest molecule can influence the reaction pathway. As a first step towards molecular factories, we will investigate multi-catalyst tandem reactions utilizing two or more nano-reaction chambers. Natural enzymes will be combined with man-made nano-reaction chambers to obtain novel reactivity. |
The objective of this project is to move beyond primary systems to maximize complexity and cumulate emergent properties that are a) significant (conceptual or practical), b) absent in the individual components, and c) inaccessible otherwise. |
Ongoing projects focus on the development of orthogonal dynamic covalent bonds for advanced systems interfacing; current emphasis is on boronic esters from bioadhesives. A second specific objective is the creation of artificial enzymes that operate with interactions that are new-to-nature; current emphasis is on the interfacing of anion-π interactions and streptavidin mutant libraries. A third specific objective focuses on disulfide exchange chemistry on cell surfaces to interface living cells with functional systems such as protein complexes involved in gene editing, artificial metalloenzymes for metabolic engineering, and liposomes or polymersomes as artificial organelles. In this project, emphasis is exclusively on added value from collaborations within the network of this NCCR, i.e., research that could not be realized without this NCCR. Scientific Highlights
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Forces matter in molecular systems engineering. Engineered microsystems, when exposed to living tissues, will be strained by mechanical forces, for example when exposed to shear forces, or when colliding with blood vessel walls or grabbed by our immune cells. Since one central goal of this NCCR is to engineer a new generation of engineered micro-or nanosystems that can interfere at the functional level with cells and tissues in vivo, we are particularly interested in asking how their mechanical characteristics have to be designed to best serve their functions. While major efforts in the past went into the chemical design of drug delivery systems, the mechanobiology of cells and tissues, and how this co-regulates uptake and other cell functions, has been mostly ignored in their design. |
To learn from nature how to best engineer molecular factories that survive the strenuous conditions found in living organisms, we currently investigate the methods by which cellular microorganisms interact with biological systems, and quantify the forces involved and how this knowledge can be utilized to engineer synthetic micro and nano-objects. Once microbes enter the body, for example, they often adhere to tissue surfaces or fibers. It is thus not sufficient that macrophages and other immune cells recognize just the biochemical signature of molecular factories. Either one can design these molecular factories to be “invisible” to the immune system. Alternatively, if the goal is to have these factories enter cells, it is important that cells can apply sufficient forces to rupture the adhesive contacts by which these objects hold on to tissue surfaces, and that the cells can subsequently form a phagocytotic cup around the objects which then allows their phagocytosis. One central goal of us is thus to develop assays that allow us to test for mechanical processes by which cells of different organs as well as circulating cells interact with microbes and how this can best mimicked by engineered biomimetic objects. An associated goal is to develop cellular read out systems to learn how these mechanical processes affect cell signaling responses. Since we anticipate a broad range of medical applications made possible by the engineered microsystems of this NCCR, our goals include to ask how we need to learn (1) how sessile cells (e.g. fibroblasts) and circulating immune cells (e.g. macrophages) recognize and interact with the surface chemistry, shape and physical properties of the microsystems, and (2) how to (re)engineer such containers either to promote, for example, the uptake by cells for longterm applications in immune therapy, or to reduce the uptake by cells for other applications. This includes investigating how size and shape affect uptake by macrophages and to analyze spatial-temporal evolution of the forces involved, and how the mechanical forces cells apply to such engineered objects impede or steer cellular signaling. Read more:
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Contrary to the macroscopic world, the engineering of integrated systems at molecular level imposes a complex scenario of requirements, especially for assembling biotic and abiotic components with the dual aim; to preserve the specific functionality of each entity, and to induce new functionalities and properties serving a final desired application. |
A variety of molecular entities such as membranes, self-adapting vesicles, and biological molecules (enzymes, proteins, mimics) can be combined to design hybrid supramolecular assemblies with multifunctionality and emerging properties. Our aim is to organize nanometer sized assemblies, such as polymer vesicles, into clusters/networks with controllable size and tunable biological properties. We rely on molecular recognition interactions, as for example on DNA hybridization to interconnect vesicles. In addition, we use the non-hybridized single strand-DNA as a molecular anchor to bind the clusters selectively to the cell surface. Each polymer vesicle can be tailored to include biologically relevant enzymes, substrates, active molecules and membrane pores to allow communication between the compartments and cascade reactions inside. Such clusters/networks will be evaluated in vitro and in vivo to establish their interactions with cells, their stability and functionality for desired bio-applications. Collaborations within NCCR with other research groups with competences in polymer chemistry, inorganic chemistry, molecular biology, metabolic engineering and characterization methods for complex systems/reactions will be essential for a complete design and characterization of such hybrid systems. Our platform represents an important step for development of molecular factories because it has a high potential for biological applications, such as protein therapy or active templates for regenerative medicine. |
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| Projektleitung | Mitarbeiter | [grouplink] |
|---|---|---|
| Michael Nash | N.N. | Nash group @UniBas |
| Emanuel Lörtscher | N.N. | Lörtscher group @IBM |
| Ivan Martin | N.N. | Martin group @UniBas |
| Konrad Tiefenbacher | N.N. | Tiefenbacher group @UniBas |
| Stefan Matile | N.N. | Matile group @UniGe |
| Barbara Treutlein | N.N. | Treutlein group @D-BSSE |
| Viola Vogel | N.N. | Vogel group @ETHZ |
| Cornelia G. Palivan | N.N. | Palivan group @UniBas |
To reduce the complexity of multi-component reactions, individual reaction sites are physically separated into compartments, which are interlinked by microfluidics for mass-flow as well as electrical, optical and electrochemical exchange (Fig. 1A). Electrically contacted molecular monolayers (Fig. 1B) can exert molecular-intrinsic functionalities to be used for local sensing, energy-generation, storage or release sites (Fig. 1C).
Electrode surfaces can be site-selectively functionalized by immobilizing a precursor compound with an electrochemically active protecting group that undergoes an irreversible chemical cleavage after reductive activation (Fig. 2A). This method allows chips with buried microfluidic channels and enclosed compartments to be efficiently functionalized by simple click-chemistry under mild conditions and with any desired functionalities (Fig. 2B).
Selective molecular binding between an immobilized receptor and an analyte change the dielectric environment of nano-structured surfaces (Fig. 3A). Their optical and electrical properties can be read-out by various modalities (Fig. 3B) leading to local sensing capabilities within a reaction compartment, a crucial requirement for feedback control in a molecular factory. For that task, we develop chemical anchoring concepts to adhere to both crystalline and amorphous silicon surfaces.
Nanoscale separation of electrodes in semiconductor parallel-plate devices provides oriented electrical fields with amplitudes exceeding 109 V/m, giving rise to novel field-assisted chemistry. Combined with a directed assembly of molecular compounds on appropriated electrodes, such external fields can be used to control reactivity (Fig. 4A) or selectivity (Fig. 4B) in organic synthesis or to introduce novel synthetic processes.
The site-selective functionalization of large arrays of reaction compartments on a silicon platform by catalytically active, immobilized compounds - in conjunction with local reaction control and nanoscale constraints - paves the way to synthesize large product libraries from a common starting material by combinatorial, divergent heterogeneous catalysis (Fig. 5). This effort finally aims at screening new synthetic pathways for drug discovery.