Nonequilibrium phases of a biomolecular condensate facilitated by enzyme activity.

Biomolecular condensates represent a frontier in cellular organization, existing as dynamic macromolecular structures driven out of equilibrium by active cellular processes. Here we explore active mechanisms of condensate regulation by examining the interplay between DEAD-box helicase activity and RNA base-pairing interactions within a reconstituted ribonucleoprotein condensate. We demonstrate that the ATP-dependent activity of a DEAD-box helicase-a key class of enzymes in condensate regulation-acts as a nonequilibrium driver of condensate properties through the continuous remodeling of RNA interactions. By combining the LAF-1 DEAD-box helicase with a designer RNA hairpin concatemer, we unveil a complex landscape of dynamic behaviors, including time-dependent alterations in RNA partitioning, evolving condensate morphologies, and shifting condensate dynamics. Importantly, we reveal an antagonistic relationship between RNA secondary structure and helicase activity which enables an initially homogeneous nonequilibrium state. By elucidating these nonequilibrium mechanisms, we gain a deeper understanding of cellular organization and expand the potential for active synthetic condensate systems.