Modular RNA motifs for orthogonal phase-separated compartments

Phase separation of biomacromolecules into immiscible membrane-less organelles contributes to regulating distinct biochemical functions. However, re-enacting and controlling such orthogonality remains a challenge. In this work, we constructed pure-RNA condensates from single-stranded motifs (monomers) and verified their ability to co-exist as orthogonal compartments that were tracked with the addition of multicolor fluorogenic RNA aptamers. The RNA monomers, which fold into star-shaped structures, are designed to interact through HIV-typed kissing loops and generate different droplet morphologies based on the sequence designs. Furthermore, we demonstrated that these single-stranded nanostars can form droplet-like condensates cotranscriptionally in vitro in physiological conditions. Our work showcases strategies to build a highly programmable library of orthogonal RNA assemblies facilitating the understanding of the spatial organization of bioprocess and offers a potential tool to engineer artificial organelles within living cells.