The ERC project ENSYNC (From engineering to evolution of synthetic cells with RNA origami) aims to pioneer the construction of a synthetic cell from the bottom-up, using RNA as the key functional building block. The project’s central goal is to establish a system where the synthetic cell's physical properties (phenotype) are directly linked to its genetic information (genotype), laying the groundwork for it to undergo Darwinian evolution.
During this reporting period, we have made significant foundational progress and achieved several key breakthroughs:
Genetically Encoded RNA Hardware inside Synthetic Cells: A major achievement was the first-ever de novo expression of complex, 3D RNA origami nanostructures inside cell-sized lipid vesicles (GUVs). As published in Nature Nanotechnology, we successfully designed a DNA template that, when transcribed inside the vesicle, produces RNA tiles that self-assemble into a micrometre-scale, genetically encoded cytoskeleton.
Genotype-Phenotype Link: We demonstrated that rational mutations in the DNA template (genotype) lead to predictable changes in the final RNA structure (phenotype, e.g. from nanotubes to rings), validating the core hypothesis for evolvability.
Functional Readout: We further functionalized this RNA cytoskeleton to bind the vesicle's inner membrane, forming a cortex that actively deforms the synthetic cell. This provides a direct proof-of-concept for the project's goal of using genetically encoded RNA structures to mechanically control cell shape, a foundational principle for synthetic cell division.
Sustained, Out-of-Equilibrium Activity: To power this in-vesicle synthesis, we successfully established a "feeding bath" system (also detailed in Nature Nanotechnology). By incorporating α-haemolysin pores into the GUV membrane, we enabled the continuous influx of nucleotide "fuel" from the outside environment. This allowed for sustained, out-of-equilibrium transcription and assembly, transforming the synthetic cell from a static compartment into an active, continuously running system.
Development of Critical RNA Components: In parallel, we have successfully designed and validated another critical piece of RNA hardware: a transmembrane RNA nanopore. This component is designed to be genetically encoded and provide the synthetic cell with its own "gateway" to the environment, a key step towards autonomy. Functional validation via dye influx assays and structural characterization by cryo-EM has been completed, and a manuscript is in preparation.
Enabling Technology for RNA Design: To manage the high complexity of these designs and accelerate future work, we developed and published "PyFuRNAce" (Nature Communications). This is the first open-source, user-friendly, and integrated software engine for co-transcriptional RNA origami design. This tool is a key enabling technology for the entire field and provides the essential computational backend for our project's next phase: high-throughput design and directed evolution.
