Intracellular protein condensates form and dissolve through a balance of interactions that often involve RNA. Some proteins use defined RNA-binding domains, while others rely on electrostatic interactions mediated by intrinsically disordered regions. The dynamic and heterogeneous nature of condensates makes it difficult to resolve the molecular specificity and stoichiometry of RNA–protein interactions using conventional ensemble approaches. The preprint reports an application of single-molecule mass measurements to address these limitations and to monitor molecular assembly states during condensate phase transitions.
The authors used mass photometry, a single-molecule mass measurement technique, to capture RNA–protein interactions in phase-separated protein systems. Mass photometry measures the mass of individual particles in solution, enabling direct observation of assembly states without relying on ensemble averaging. The study positions mass photometry as a complementary method for distinguishing interaction modes that underlie phase separation in RNA-containing systems.
The authors applied mass photometry to investigate how RNA concentration influences the phase behavior of tau condensates. They report that increasing RNA concentration—conditions that promote phase re-entry of tau—leads to RNA-mediated tau multimerization rather than only charge neutralization. According to the measurements described, each tau monomer within the observed multimers binds a linear RNA segment of approximately 30 nucleotides. These single-molecule mass observations indicate a shift in interaction mode as RNA concentration changes: from interactions consistent with coacervation toward formation of defined RNA–protein complexes that drive re-entry into the condensed state.
To corroborate the mass photometry data, the authors used solution NMR and native mass spectrometry. These orthogonal methods confirmed the formation of stable complexes between RNA and specific regions of tau. The interactions were localized to the basic proline-rich and repeat domains of tau, which the authors report as having a net charge of −29. Together, these techniques support the interpretation that RNA forms defined complexes with tau domains under conditions that favor phase re-entry, rather than simply neutralizing charge to drive phase separation.
A central conclusion is that mass photometry can distinguish two mechanistic scenarios in RNA-mediated phase behavior. One scenario is coacervation driven by charge neutralization, in which electrostatic screening drives condensate formation without formation of stable stoichiometric complexes. The alternative scenario demonstrated here is complex formation, where RNA binds tau in defined assemblies that mediate phase re-entry. The single-molecule mass readout allowed the authors to resolve multimerization and RNA-binding stoichiometry that are not readily separable by bulk measurements.
The study demonstrates that single-molecule mass measurements using mass photometry can capture shifting RNA–protein interactions during condensate phase transitions. Applied to tau, this approach revealed RNA-mediated multimerization and a repeatable binding stoichiometry of about 30 nucleotides per tau monomer, supported by solution NMR and native mass spectrometry. The authors present mass photometry as a complementary technique for interrogating RNA-mediated phase separation, capable of distinguishing charge-neutralization-driven coacervation from complex-driven phase re-entry.
Limitations and caveats reported in the source: this work is presented as a preprint and has not been peer reviewed. The preprint provides experimental observations and corroborating biophysical data, but full methodological and quantitative details are contained in the manuscript; readers should consult the full preprint for experimental specifics and supporting figures.
The authors declare that one author (MFH) is an employee of Refeyn Ltd. Funding sources listed in the preprint include Cancerfonden, the Swedish Research Council (VR), the Knut and Alice Wallenberg Foundation, the Swedish Society for Medical Research, the Swedish Alzheimer Foundation, the Swedish Brain Foundation, CIMED, and Karolinska Institutet. The article is a bioRxiv preprint and has not been certified by peer review.