Single-molecule mass measurements uncover shifting RNA interactions during condensate phase transitions

RNA interactions are a key contributor to the formation and disassembly of intracellular protein condensates. Although some proteins utilize specific RNA-binding domains, these processes can also be mediated by charge interactions with intrinsically disordered regions. Due to the dynamic nature of these systems, investigating the underlying specificity and stoichiometry remains challenging. Here, we demonstrate that single-molecule mass measurements with mass photometry can capture RNA-protein interactions in phase-separated protein systems. Using the approach to investigate RNA-mediated phase shifts of tau condensates, we find that increasing the RNA concentration, which promotes phase re-entry, results in RNA-mediated tau multimerization, where each tau monomer binds a linear RNA sequence of approximately 30 nucleotides. Solution NMR and native mass spectrometry confirm the formation of stable complexes between RNA and the basic proline-rich and repeat domains of tau, which have a net charge of -29. Our findings demonstrate that mass photometry can distinguish between charge neutralization, which drives coacervation, and complex formation, which mediates phase re-entry, making it a highly complementary tool for the study of RNA-mediated phase separation.