Living organisms have acquired new functionalities by uptake and integration of species to create symbiotic life-forms. This process of endosymbiosis has intrigued scientists over the years, albeit mostly from an evolution biology perspective. With the advance of chemical and synthetic biology, our ability to create molecular-life-like systems has increased tremendously, which enables us to build cell and organelle-like structures. However, these advances have not been taken to a level to study comprehensively if endosymbiosis can be applied to non-living systems or to integrate living with non-living matter. The aim of the research described in the ARTISYM proposal is to establish the field of artificial endosymbiosis. Two lines of research will be followed. First, we will incorporate artificial organelles in living cells to design hybrid cells with acquired functionality. This investigation is scientifically of great interest, as it will show us how to introduce novel compartmentalized pathways into living organisms. It also serves an important societal goal, as with these compartments dysfunctional cellular processes can be corrected. We will follow both a transient and a permanent approach. With the transient route biodegradable nanoreactors are introduced to supply living cells temporarily with novel function. Functionality is permanently introduced using genetic engineering to express protein-based nanoreactors in living cells, or via organelle transplantation of healthy mitochondria in diseased living cells. Secondly I aim to create artificial cells with the ability to perform endosymbiosis; the uptake and presence of artificial organelles in synthetic vesicles allows them to dynamically respond to their environment. Responses that are envisaged are shape changes, motility, and growth and division. Furthermore, the incorporation of natural organelles in liposomes provides biocatalytic cascades with the necessary cofactors to function in an artificial cell

In the last decade, super-resolution microscopy techniques have emerged as powerful quantitative tools for biology. They have capabilities to visualize single molecules at the nanoscale opening the door to study biological processes at a level not accessible before. In the ERC StG NANOSTORM we showed the potential of these techniques providing new fundamental knowledge on the mechanism and design of new targeted therapies. However, some of the methods we developed have the potential to be translated into applied clinical diagnostic tools. In NANODIAGNOSTIC, we would offer a proof-of-concept of the application of super resolution microscopy and single-molecule imaging for cancer diagnostic, with a focus on patients stratification for immunotherapy. Novel advances in immunotherapies have brought the development of immune checkpoint inhibitors (ICI) that re-activate the immune system against the tumor. Despite the high success of these therapies there is one main challenge: they are only effective on a limited portion of patients and current diagnostic approaches are not capable of stratifying patient successfully. NANODIAGNOSTIC will translate advance optical methods from an academic setting to the clinic and holds a great potential to provide new diagnostic methods to improve the outcome of immunotherapy.

Many supramolecular systems have been inspired by nature, but the number of supramolecular systems that are truly functional in water at the low concentrations required for biomolecular studies are very limited. Cucurbiturils are one of a few select supramolecular systems that show great promise for the modulation of protein assemblies in biologically relevant media, but they require better means to control homo- and heterodimerization. In order to effect strong and selective heterodimerization I will design and synthesize a wide range of complementary guest pairs, using chemical and electronic concepts such as π-π stacking and electronic donor−acceptor pairs. After testing these on the cucurbituril host-guest system, they will be assessed on heterodimeric protein assemblies such as split luciferase. As many biological processes require multimeric protein assemblies, I will develop novel supramolecular constructs to gain control over the formation of such assemblies. By constructing protein-coupled cucurbiturils and developing novel double cucurbituril systems, trimeric and tetrameric protein assemblies will be assessable. Development of these advanced supramolecular tools is crucial in order to access synthetic signaling platforms with potential for molecular diagnostics.