A DNA Segregation Module for Synthetic Cells.

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells.

Genotype-phenotype mapping with polyominos made from DNA origami tiles.

The correlation between genetic information and characteristics of a living cell-its genotype and its phenotype-constitutes the basis of genetics. Here, we experimentally realize a primitive form of genotype-phenotype mapping with DNA origami. The DNA origami can polymerize into two-dimensional lattices (phenotype) via blunt-end stacking facilitated by edge staples at the seam of the planar DNA origami. There are 80 binding positions for edge staples, which allow us to translate an 80-bit long binary code (genotype) onto the DNA origami. The presence of an edge staple thus corresponds to a "1" and its absence to a "0." The interactions of our DNA-based system can be reproduced by a polyomino model. Polyomino growth simulations qualitatively reproduce our experimental results. We show that not only the absolute number of base stacks but also their sequence position determine the cluster size and correlation length of the orientation of single DNA origami within the cluster. Importantly, the mutation of a few bits can result in major morphology changes of the DNA origami cluster, while more often, major sequence changes have no impact. Our experimental realization of a correlation between binary information ("genotype") and cluster morphology ("phenotype") thus reproduces key properties of genotype-phenotype maps known from living systems.

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling.

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV-based communication and how protein-based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle-induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV-membranes. By bottom-up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV-like vesicles, increases their signalling potential by more than 100-fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV-presented proteins. By this the authors propose vesicle-triggered local clustering of membrane receptors as the principle structural mechanism underlying EV-based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter-protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV-based intercellular signalling.

Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.