Projekte

We engineer synthetic cellular memory platforms that enable the reconstruction of cellular histories and can be applied as living diagnostics.

A fundamental challenge in biology is to understand how cells function and integrate complex molecular information to perform different behaviors. For example, the differentiation of a stem cell into two daughter cells with distinct identities or the transformation of a normal cell into a cancer cell. This challenge has motivated the creation of numerous technologies facilitating detailed intracellular observations at the level of DNA, RNA, protein, and metabolites. Despite the power of these approaches, they generally require destructive methods and therefore observations are limited to a few snapshots in time or select asynchronous cellular processes. One provocative solution to this is to introduce DNA writing and molecular recording platforms within cells that enable the encoding, storage, and retrieval of molecular information.

Towards the goal of continuously recording molecular events within cells, my laboratory is developing and applying Record-seq, a ‘transcriptional recording’ platform that employs CRISPR spacer acquisition from RNA to capture and convert intracellular RNAs into DNA, permanently storing transcriptional information in the DNA of living cells. The newly acquired sequences serve as transcriptional records, which are retrievable via deep sequencing and can be leverage to reconstruct cellular histories. This technology provides an entirely new mode of interrogating dynamic biological and physiological processes and opens up numerous avenues for future work in engineering cellular systems.

The chemical and physical processes enabling the transformation of matter in living systems is regulated by complex feedback loops. Such tightly cross-regulated processes enable highly complex reaction cascades as well as transport and exchange mechanisms that have not yet been achieved in synthetic systems. The project „Nanopores as Solid-State Approach to Interlinked Reaction Compartments" strives to simulate isolated feedback mechanisms to investigate the underlying regulation mechanisms in detail and to identify the parameters governing the system.

To fabricate such interlinked reaction compartments, we aim at fabricating nanopore devices based on a solid-state approach using top-down fabrication techniques. Self-assembled monolayers (SAMs) of functional molecular building blocks are physically separated but remain addressable by electrical, optical or electrochemical means. The SAMs are highly oriented which enables correlations between chemical structure and electronic as well as ionic transport properties in single-molecular junctions to be studied. 

Preliminary studies will allow us to mimic a biological response and to investigate isolated feedback mechanisms in detail. Additionally, from a materials point of view, the resulting oligomers may be interesting. Their physical properties are length-dependent and, thanks to a feedback mechanism, their length-distribution may become tunable by specific parameters dictated by the system, leading to a new size-control approach with wide potential applications in material science. Modular systems can easily be expanded with additional functionalities. For example redox-­dependent chromophores that will facilitate the investigation of the systems dynamics as it can be investigated by optical microscopy.

The project is not only geared towards investigating feedback mechanisms across vesicle membranes but also towards integrating vesicles as molecular factories. With such vesicles, molecular devices enabling photo-induced charge separation across the vesicle membrane will be studied. In a later stage of the project, we envisage electrochemical interconnecting of different functionalized vesicles to build up gradients of chemical potentials. 

Building autonomous synthetic organelles and cells with a defined function using a repertoire of functional modules (toolkit) and containing inside a minimal metabolism for survival, represents the ultimate goal of this project group.

Such complex processors will open a wide variety of possibilities ranging from environmental to medical applications. One of the most important challenges will be to provide a large repertoire of engineered and modular biomolecular-transport and -energy conversion systems for assembly of nanoreactors with diverse functionalities in lipid bilayers and block copolymers.

Initially, modules will include light-­driven proton pumps and proton-driven solute transporters in the membrane, and metabolizing enzymes inside the container. Next, more complex powering systems will be explored such as combinations of light-­driven proton pumps with sodium/proton antiporters with the objective: to energise sodium-driven solute transporters. This will significantly increase the repertoire and specificity of translocating modules.

The availability of numerous, highly specialized membrane proteins in milligram amounts offers the unique opportunity to use them as building blocks and toolkit to assemble molecular factories in the form of nanoreactors and functional surfaces using bottom-up approaches.

This project group has a strong expertise and knowledge in biochemistry, function and structure of membrane proteins. Furthermore, the group already possesses a significant number of recombinant transport proteins for different solutes such as peptides, sugars, amino acids and antibiotics that can be used as modules for engineering and assembly of nanoreactors.

Molecular Systems Engineering (MSE) incarnates a novel approach to clinical innovation that considerably expands the toolbox of molecular sciences and healthcare both theoretically and technically. The prospects of this emerging field to bring about scientific and clinical innovation crucially depend on proactively addressing potential ethical and regulatory bottlenecks.

Such issues include three major domains, that is: issues associated with society’s appraisal of the novel bio-technological characteristics of engineered molecular systems; the ethical and legal aspects linked to the clinical translation of MSE into healthcare applications; and the development of appropriate regulatory standards for the assessment of MSE applications by regulatory agencies.

To address those issues, this project brings new empirical research and analytical competences on ethics and regulatory issues of MSE technologies. Thanks to considerable experience and reputation in the field of bioethics and health policy, the ETH Zürich’s Health Ethics and Policy lab led by the PIs Effy Vayena, and Alessandro Blasimme will ensure dedicated research on all of the above issues.

Normative and empirical research on the ethics of MSE will result in specific ethical guidelines to guide the long-term development of the field in the future. As far as regulatory aspects are concerned, relevant national and international stakeholders – including regulators – will be engaged and a regulatory roadmap for MSE will be developed.

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.