Phase separation kinetics of biomolecular condensates under passive and active conditions

We investigate the growth kinetics of condensates formed by cationic peptides (RRASLRRASL) and poly uracil (polyU) RNAs, serving as an ideal model system for membrane-less organelles. Employing absorbance measurements at 500 nm, the critical peptide concentration is estimated to be 400 µM at a fixed polyU concentration (0.5 g/L) in a physiological buffer (50 mM HEPES pH 7.4) at 37°C [Aumiller et al, Nature Chem. 2016]. Confocal microscopy images of fluorescently labeled peptide/polyU condensate at this concentration display a spherical morphology with diameters ranging from 1 to 5 µm.

Time-resolved small-angle X-ray scattering (TR-SAXS) experiments with a stopped-flow device revealed that the condensate size increased rapidly, reaching several hundred nanometers in less than 400 ms. The condensate radius R scales with time t as R ∼ t^α with α = 0.31, which is consistent with the expected value of ⅓ for a standard coalescence or ripening process.

We further investigated the dephosphorylation of double-phosphorylated peptides by Lambda Protein Phosphatase (LPP), which dephosphorylates the serine residues of the peptide, thus driving condensation. TR-SAXS curves indicated that condensates began to form at around 20 min with their radius expanding from 100 to 900 nm by around 40 min. The formation of condensates is significantly slower with phosphorylated peptides compared to the unphosphorylated form, and the time evolution of size distribution is currently investigated by confocal microscopy.