Fig. 1: R3C ligase forms phase-separated coacervate compartments with simple cationic oligopeptides.
a, Schematic representation of RNA–peptide compartment formation. The coacervate is shown as a fluorescent microscopy image with Cy5-labelled RNA. b, Schematic representation of the RNA-ligation reaction and experimental validation of R3C ligase product formation by gel electrophoresis. Reactions were carried out in 5 mM MgCl2 and 10 mM Tris pH 7.5 and contained 5 µM E, 0.1 µM Cy5-S1 and 6 µM S2. The band shift of Cy5 5′-labelled S1-RNA is indicative of ligation. c, Turbidity plots indicate the formation of stable coacervates over a broad peptide concentration regime for various Rn and longer (RGG)n peptides, whereas for Kn peptides other than K9 coacervation was found to be concentration sensitive. Data are shown as mean ± 68% confidence interval; grey open circles indicate independent experiments; n = 3 for all peptides. %T describes the percentage of transmittance. All turbidity assays were performed using 5 µM E and 6 µM S2, a total of 438 µM of nucleotide. d, Electrophoretic mobility shift assays indicative of RNA–peptide complex formation using FAM-labelled RNA and SyproRed peptide staining. Samples contained 5 µM E, 6 µM S2 and 0.1 µM 6-FAM E in 5 mM MgCl2 and 10 mM Tris pH 7.5. Electrophoresis was carried at room temperature. Red boxes indicate accumulation of RNA in the wells. e, Turbidity plot for R9 showing a sigmoidal transition between soluble RNA–peptide complexes and coacervates. Data are shown as mean ± 68% confidence interval; grey open circles indicate independent experiments; n = 3. The plot shows three distinct regimes: soluble complexes (I), onset of coacervation (II) and appearance of peptide-saturated coacervates (III).
