Developing synthetic RNA organelles for spatiotemporal separation, control, and monitoring in living cells

Project summary Our understanding of biology is being transformed by the discovery and characterization of organelles arising from the spontaneous phase separation of proteins and RNA, in the absence of a lipid membrane. These membraneless organelles, also called condensates, occur at different cellular locations and can appear as solids, gels, or liquids. Dozens of distinct biomolecular

condensates are associated with pathways that regulate genes, stress response, and, and their function depends on the types of molecules they recruit from the cellular environment. This has spurred the interest toward developing means to harness condensation by building artificial condensates, that could be used for separating molecules in vitro and as organelles inside living

cells. Most efforts in this direction rely on engineered proteins that include disordered domains: this approach however is hampered by difficulties in building proteins presenting well-defined interactions, minimal promiscuity, and limited side effects. These challenges can be addressed by adopting engineered RNA, rather than proteins, as a building block for artificial condensates,

because specific RNA-RNA interactions are easy to program, and RNA is a molecule easily portable across organisms presenting low toxicity. This project aims to develop a new class of artificial condensates by taking advantage of nanostructured RNA. We will build RNA condensates capitalizing on our recent discovery that

star-shaped RNA motifs (nanostars), comprising a single molecule of RNA, can produce dense RNA droplets in cell-free samples and in living cells. By bridging concepts in phase separation science and RNA nanotechnology, our project will establish RNA nanostars as a platform to build customizable RNA organelles through different research focus areas aimed at: (1) developing

methods for sequence and structure optimization, leading to condensates with desired thermodynamic and biophysical properties, and with specific affinity for separating guest molecules; (2) gaining control over the location, kinetics, and composition of RNA organelles forming inside cells; (3) establishing means to build RNA condensates that can sense and

respond to molecular signals, and explore their usefulness as sensing and imaging tools. By providing a tunable platform to control the spatial and temporal distribution of target molecules within living cells, our synthetic organelles will serve as a powerful tool toward achieving control of gene expression and biosensing.