Programmable and Switchable RNA Scaffolds for Synthetic Condensate Engineering in Mammalian Cells

The ability to engineer synthetic biomolecular condensates in living cells offers new opportunities to control intracellular organization, yet robust and programmable RNA-based systems have remained limited. Here, we introduce genetically encoded, modular platforms that generate RNA-driven condensates using nanostar-derived scaffolds. Systematic comparison of repeat-based and <i>de novo</i> designs identified nanostar variants that reliably assemble nuclear condensates in mammalian cells. Unexpectedly, condensate formation in cells is governed primarily by double-stranded RNA stems that recruit endogenous RNA-binding proteins, rather than by the kissing-loop interactions that drive assembly <i>in vitro</i>. This mechanistic shift highlights the divergence between cellular and <i>in vitro</i> environments and accounts for the limited orthogonality among scaffolds. Sequence refinement to reduce nonspecific pairing improved homotypic assembly and enhanced orthogonality. We further demonstrated functional compartmentalization by recruiting protein and RNA clients to modulate their stability and activity, and we incorporated an acyclovir-responsive allosteric switch to achieve reversible, small-molecule control of condensation. Together, this work establishes a versatile RNA-based toolkit for constructing programmable cellular compartments, advancing strategies for controlling RNA-protein organization and enabling new biosensing and therapeutic applications.