Figure 6.
Experimental evidence for the stoichiometry-dependent liquid-liquid phase separation of RGG-polyU mixtures
(A) A state diagram indicating the RGG (FUSRGG3) and polyU concentrations at which condensates form (red-filled circles) and the concentrations at which the mixture forms a homogeneous state (blue-filled circles). The solid black line is a guide to the eye. The red shaded region marks the LLPS regime. Inset shows a fluorescence microscopy image of RGG-polyU condensates formed at an RGG concentration of 0.33 mg/mL and polyU-to-RGG ratio of 0.75 (w/w). The buffer contains 25 mM Tris-HCl (pH 7.5), 125 mM NaCl, and 20 mM DTT. Scale bar represents 10 micron.
(B) A state diagram showing the effect of increasing ionic strength of the buffer on the LLPS region of RGG-polyU mixtures. Higher salt concentration leads to the shrinkage of the LLPS region in the state diagram because of weaker intermolecular interactions (see Figure S1).
(C) The turbidity of RGG-polyU mixtures as a function of RGG-to-polyU ratio (w/w) is shown for several salt concentrations (NaCl). The shaded regions indicate the LLPS condition (turbidity >0.25) for the lowest and highest salt concentrations used in this study and are drawn as a guide to the eye. The black arrow marks the shrinkage of the LLPS window upon increasing salt concentration. The sample was prepared by fixing RGG concentration to 0.347 mg/mL and varying polyU concentration in a 25 mM Tris-HCl (pH 7.5) buffer with 20 mM DTT and the desired NaCl concentration.
(D) Similar data to (C) but at different crowding conditions. The arrow indicates the enlargement of the LLPS window upon increasing crowder concentration (PEG8000), indicating more favorable LLPS at high crowding conditions. RGG concentration is kept at 0.347 mg/mL, and polyU concentration is varied. The sample was prepared by fixing RGG concentration to 0.347 mg/mL and varying polyU concentration in a 25 mM Tris-HCl (pH 7.5) buffer with 150 mM NaCl, 20 mM DTT, and the desired PEG concentration (w/v).