Projekte

In this project we aim to use visible light to drive electrons across a membrane and to accumulate charges in such a way that NAD⁺ (nicotinamide adenosine dinucleotide) can be converted to NADH. The latter can then fuel enzymatic cascade reactions which ultimately lead to value-added products that are synthesized in the closed compartment of a molecular factory. The use of vesicles made from block copolymer membranes that make up the closed compartment offers the advantage that the enzymatic chemistry can be performed in a protected environment and that oxidation and reduction products resulting from photochemistry can be spatially separated from one another.

The photophysical and photochemical aspects of this project are addressed by the Wenger group while the enzyme chemistry is taken care of by the Ward group. Vesicles built from block copolymers will be made in collaboration with the groups of Meier and Palivan.

In the first phase of the project, strategies for photodriven electron accumulation are explored in suitable model compounds. For example, covalently linked Ru(bpy)₃²⁺ (bpy = 2,2’-bipyridine) – naphthalene diimide (NDI) systems are investigated with a view to reducing NDI to NDI^- and finally NDI^2- in the course of photoirradiation of the Ru(bpy)₃²⁺ photosensitizer in presence of sacrificial or non-sacrificial electron donors. Once the challenge of achieving efficient electron accumulation has been tackled, it will be possible to perform light-driven two-electron reduction of suitable small molecule catalysts which are able to reduce NAD⁺ to NADH.

In parallel, the Wenger group explores the possibility of obtaining other fuels by means of photochemistry, for example formate from CO₂. Formate can act as an electron source for various enzymatic reactions hence this molecule can potentially be used to initiate enzyme cascades. Close interaction between the Wenger and Ward groups is key for this purpose. 

Furthermore, strategies for the oriented incorporation of molecular wires into block copolymer membranes are explored jointly between the Palivan and Wenger groups. In order to obtain net electron transfer from the outside to the inside of the molecular factories made from vesicles, it will be essential to orient donor-bridge-acceptor systems properly in the membrane. 

Possible long-term perspectives include the incorporation of proton pumps into the vesicles in order to transport protons across the membranes, in addition to electrons. This avenue will be explored jointly with the group of Fotiadis. An addition possible avenue is the use of artificial metalloenzymes in the enzyme cascades that rely on engineered anion-π catalysis, as implemented jointly be the groups of Matile and Ward.

Strategies to integrate and exploit artificial metalloenzymes within mammalian cells for biomedical applications.

With synthetic biology applications in mind, attempts to exploit organometallic catalysts within a cellular environment have been met with limited success. Thanks to their know-how in organometallic- and bioinorganic chemistry with an emphasis on catalysis, this group has pioneered the field of artificial metalloenzymes (ArMs) based on the non-covalent incorporation of a catalytically competent organometallic cofactor within a host protein. The resulting hybrid catalysts display features which are reminiscent of both homogeneous- and enzymatic catalysts. 

With biomedical applications in mind, the main challenge to be addressed within the NCCR Molecular Systems Engineering is the integration of ArMs within mammalian cells. The ultimate goal of this effort is to complement natural enzymes for the on-site production or uncaging of drugs and for diagnosis purposes. The bio-orthogonality of some of the ArMs developed within the group allows to complement the natural reaction repertoire. To integrate artificial metalloenzymes within mammalian cells, the following strategies will be investigated: i) encapsulation within polymerosomes or ii) incorporation of cell-penetrating disulfides on ArMs. Alternatively host proteins overexpressed on the cell surface of diseased cells will be exploited to accumulate organometallic cofactors which can uncage drugs and accumulate drugs where needed.

Benefitting from synergies and the unique expertise of various projects within the NCCR Molecular Systems Engineering, the focus will be on the design of cellular diagnosis and therapeutic production factories that detect and correct physiological disorders.

In modern medicine, diagnosis of disorders kick-off therapeutic interventions and early-stage discovery of pathologies significantly improves therapeutic success. However, most disorders will only be diagnosed when discomfort urges a patient to seek medical advice. In these cases treatment may be too late.

Tumour markers, immune response proteins and pathology-associated metabolites are monitored for diagnosis and therapy management by quantitative analysis of blood samples or biopsies. This requires medical intervention that is typically initiated when the patient has symptoms and is seeking medical advice. However, preventive medical check-ups for the prognosis of physiological disorders are not receiving enough attention.

Synthetic gene circuits that constantly monitor physiological processes, detect a pathological situation and produce diagnostic output or coordinate therapeutic interventions could change our health-care system from the standard symptom-treatment scheme to a symptom-free preventive care strategy.

Scientific Highlight

Mind-Controlled Gene Expression (read the publication in full here): The Fussenegger-Group has designed a synthetic mind-controlled gene switch that enables human brain activities and mental states to wirelessly program transgene expression in cells. This device harnesses the electric energy of a person’s brainwaves thanks to an electro-encephalogram to trigger a light-emitting diode, which remotely activates light-inducible genes (optogenetic switch) in a small implant placed in mice. This technolgy, which was selected by the Scientist as one of 2014’s “Big-Advance” in Science, may provide cell-based treatments that respond to specific mental states.

Smart implants with intrinsic biosynthetic gene expression mechanisms and compound-selective feedback control can be considered molecular factories which may provide new treatment opportunities in future gene- and cell-based therapies.

In this project, an optogenetic method to stimulate hormone production by β-cell-mimetic designer cells will be implemented in a bio-compatible implant that will be gradually equipped with complex functionalities ranging from feedback control over semi-autarkic power supply to telemetry with remote artificial intelligence systems for continuous implant control and treatment adjustment.

Reactive oxygen species (ROS) and reactive nitrogen species are produced in phagolysosomes and play central roles in our immune response, inflammation and healing. Under pro-inflammatory conditions, macrophages upregulate ROS production, which helps them to clear wound sites.

Our goal in this project is to develop tools that allow to probe and quantify ROS levels in the interior of macrophages and of other phagocytes, as well as investigate signalling changes in macrophages in response to ROS stimulation.

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 Functionalities

joint research activity with Mayor group

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).

Site-selective Functionalization of Metal and Si-surfaces

joint research activity with Mayor group

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).

Optical Sensing of Molecular Binding Interactions

joint research activity with Mayor group

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.

External Oriented Electric Field-assisted Chemistry

joint research activity with Mayor group

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.

Divergent Compound Library Synthesis by Cascaded Heterogeneous Catalysis

joint research activity with Sparr and Mayor groups

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.

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.

The project is dedicated to rational design and engineering of mammalian cells augmented with complex functionalities, for applications in non-medical biotechnology.

The two application areas include rational forward design of bioproducing mammalian cell lines, and the development of sentinel (reporter) cells for highly-informative drug discovery assays. In the former, we combine the laters tools of genetic engineering such as synthetic biology and automation, with the recent genome editing approaches and rational in situ control of the bioproduction process on a single cell level, to achieve end-to-end workflow for biomanufacturing cells without the need for screening. In the latter project, we harness the power of biomolecular computing to engineerer reporter cells that detect multiple drug effects in parallel. This dramatically increases the information content of a cell-based drug screening assay without reducing the throughput.