The kinetics of ribonucleoprotein granules

Ribonucleoprotein (RNP) granules are biomolecular condensates composed of diverse RNAs and RNA-binding proteins that play essential roles in regulating multiple aspects of RNA biology. As complex biological systems, the condensate functions of RNP granules emerge from the higher-order kinetic assembly of individual molecules. Here, we develop an in vitro single-molecule approach--termed SMART (group single-molecule assay for ribonucleoprotein granules)--that enables direct and quantitative measurements of RNP granule assembly and disassembly kinetics across nanoscale to mesoscale regimes. Using SMART, we reveal that RNP granule assembly is strongly pathway dependent. In parallel, we develop a minimal two-state mathematical model that faithfully reproduces these kinetic behaviors. This model enables quantification of multivalent interaction timescales, reconstruction of the underlying free-energy landscape governing RNP granule dynamics, and demonstrates that this assembly process is intrinsically non-equilibrium. Understanding the condensate functions of RNP granules suggests new strategies for their rational manipulation in health and disease.