Japan_IPAP - Top-down meets bottom-up: Designer membrane-less organelles from condensation of synthetic RNA nanostructure

Eukaryotic cells are characterised by the presence of internal compartments, or organelles, that by separating or bringing together specific biomolecular components can optimise processes critical for the cells to survive. Many organelles, such as the nucleus, the mitochondria and the Golgi apparatus are enclosed by lipid membranes, that have long been considered as the only means through which cells can establish internal compartments. However, in recent years, a new class of organelles has been discovered, which are not bound by a membrane. These membrane-less organelles emerge thanks to the ability of certain proteins and RNA molecules to "separate" from the rest of the cytoplasm, forming droplets due to the same physical process that causes oil droplets to separate from water. Like membrane-bound organelles, these biomolecular droplets or "condensates" play important biological roles, regulating for example, the assembly of enzymes and natural degradation of RNA.

Synthetic biology aims to re-program cells to enhance their functionalities, making them useful as therapeutic or sensing agents, and as "cell factories" for the optimised production of pharmaceutical compounds and biomaterials. Cell reprogramming is most often conducted through genetic engineering: re-writing the cell's DNA to change its behaviour.

Because of the importance that compartmentalisation plays in regulating cellular functionalities, the possibility of controlling the formation and composition of organelles would be very valuable in synthetic biology, complementing the tools of genetic engineering.

Thanks to an interdisciplinary research team from the UK and Japan, in this project, we will develop strategies to engineer the formation of non-native membrane-less organelles in cells. The new organelles will assemble from of RNA molecules that fold to form nanostructures with prescribed shape and mutual interactions. We will be able to program the RNA-organelles to capture other biomolecules, such as messenger RNA, enzymes, and other proteins, so to replicate the ability of natural organelles to spatially organise and regulate the cell's biochemical pathways. We will study the properties of the RNA organelles at first in simplified cell models: synthetic cells that mimic the properties of live cells. Later, we will move to induce the formation of the RNA organelles in live cells, specifically E. coli bacteria. Note that, as all prokaryotes, E. coli do not possess natural internal compartmentalisation, so we would be creating completely new structures within the cell!

Our findings will give synthetic biologists a new tool to fine tune the behaviour of live cells, which we argue could be particularly useful for optimising bioprocessing and biosynthesis.

Intra-cellular biomolecular condensates emerge from phase separation of proteins and nucleic acids, and are known to host diverse pathways, including RNA storage, degradation and biosynthesis, earning these objects the label of membrane-less organelles (MLOs). Besides the growing evidence supporting its biological relevance, membrane-less compartmentalisation is gaining traction as a valuable tool in bottom-up synthetic biology, e.g. in the use of hydrogel and coacervate scaffolds for synthetic cells and organelles, which have underpinned advanced functionalities including stimuli-induced payload release, predation, and spatially distributed enzymatic pathways.
Progress in controlling the structure and function of MLOs in SynCells has inspired ambitious attempts to engineer unnatural condensates in live cells. This endeavour tackles a critical bottleneck in synthetic biology, namely that of reliably re-programming internal compartmentalisation in cells. A success on this front would greatly expand our arsenal for metabolic engineering, which could have profound impact on biomanufacturing. All attempts to engineer MLOs in cells have, to date, relied on protein building blocks, but intrinsic challenges associated to protein engineering have hampered progress.
With this project will pursue an alternative route to programming membrane-less compartmentalisation in synthetic and, critically, live cells, which relies on the programmability of nucleic-acid nanotechnology. We will design genetically encoded RNA nanostructures that fold co-transcriptionally and assemble into condensates: RNA-MLOs. By tuning nanostructure design, we will be able to control the number and physical properties of RNA-MLOs, and their ability to selectively recruit proteins and nucleic acid so to host complex, spatially distributed biochemical pathways. The RNA-MLOs toolkit thus will constitute a viable solution to the challenge of rationally designing membrane-less compartments in live cells.