Switchable RNA motifs for dynamic transcriptional control of RNA condensates.

Abstract

RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA-RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA "invaders" that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of "anti-invader" strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials.