Distinct RNA Physical Microenvironments Shape Unique Properties and Functions within Biomolecular Condensates

Most of the biomolecular condensates form through multivalent interactions between proteins and RNAs. These multicomponent condensates create specialized physical microenvironments that are critical for cellular regulation. While physical microenvironments of proteins have been recently studied in condensates, the physical microenvironments of RNA remain less understood. Here, we develop a chemical method to quantitatively measure RNA physical microenvironments by integrating RNA-labeling chemistry with environmentally sensitive fluorophores and fluorescence lifetime imaging microscopy. We show that RNAs exhibit distinct micropolarity and microviscosity from proteins while remaining miscible in condensates, in contrast to protein-protein systems that demix upon differing micropolarity. This behavior may be attributed to the formation of transient RNA-protein interactions, whose status is influenced by micropolarity compatibility. We further demonstrate that micropolarity differences between RNAs and proteins correlates to RNA partitioning and selective RNA distribution among condensates. In terms of RNA function, we show that elevated RNA micropolarity is associated with ribozyme catalytic activity. Taken together, these results reveal that RNA physical microenvironments play an active role in shaping RNA organization and function within biomolecular condensates.