Abstract

Intracellular production of synthetic RNA granules by ligand-yielded multivalent enhancers

Hydrogel-like materials in cells, such as RNA granules, are believed to regulate cellular function through their unique physico-chemical properties. These materials are frequently employed as diffusion barriers at the interface of subcellular compartments. Moreover, they are often formed by a phase separation of signaling and structural molecules in order to nucleate cellular activities. While “extracellular” hydrogels have been extensively studied and elaborately reproduced in vitro, their “intracellular” counterparts have not, primarily due to technical challenges in probing, manipulating, and generating them in vivo. Here, we develop a strategy termed iPOLYMER for rationally synthesizing rapidly inducible, genetically-encoded protein-based hydrogels in vivo. This is achieved by generating a polymer network using long multivalent and flexible peptides, which are punctuated with protein domains whose association can be rapidly triggered by a chemical dimerizer. A series of biophysical characterizations in vitro and in vivo, in conjunction with computational modeling and analysis, reveal that the polymer network generated by iPOLYMER resembles a physiological hydrogel, in terms of its shape and size which behaves as a molecular sieve. In addition, we functionalize polymer networks with RNA binding motifs and synthesize artificial RNA granules that successfully sequester poly-A containing messenger RNAs. The proposed approach provides a powerful method for unraveling the key properties and functionality of hydrogel-like materials in living cells, with the potential of becoming an attractive tool for engineering cellular function through hydrogel formation.