Fig. 4: Characterization of RNA condensates as a function of overall RNA concentration.
From: Water-detected NMR allows dynamic observations of repeat-expansion RNA condensates
a, Net MT effect of biphasic (5′-CAG-3′)31, compared with the agarose meshwork (0.5%, w/v), at different concentrations of overall RNA at 250-Hz offset saturation. b, Quantification of (5′-CAG-3′)31 (150 μM), embedded in agarose (0.5% w/v) via CONDENSE-MT. Physical parameters of agarose were previously determined and held constant during fitting. Experimentally recorded MT profile is shown as a scatter plot; theoretical prediction after computational optimization is given as dashed line. The spectral area close to the H2O resonance (4.7 ppm) is shown enlarged below. Saturation amplitudes (B1) are given in Hz. Resulting physical parameters of condensed (5′-CAG-3′)31 are given within the plot. Fit deviation indicates overall deviation of experimental datapoints from the model prediction after computational optimization. c, Physical parameters of (5′-CAG-3′)31 condensates at different concentrations of total RNA, determined via CONDENSE-MT. d, Temperature-dependent microscopy of biphasic (5′-CAG-3′)31 at varying overall concentrations. Imaging occurred at 55 °C in the presence of 50 mM MgCl2. Scale bars, 2 μm. e, Schematic representation of the partition coefficient (ratio of RNA fraction in dense and dilute phase) and relative water content per unit of condensed RNA (ratio of R2,water/Pcondensate). f, Comparison of normalized population of condensed RNA (obtained via CONDENSE-MT) and normalized R2,water (corrected for R2,water = 4.13 s−1 in absence of (5′-CAG-3′)31) on variations in overall concentration of (5′-CAG-3′)31. Pcondensate and R2,water were normalized to the respective parameter at 37.5 μM total (5′-CAG-3′)31. g–i, CONDENSE-MT analysis of (5′-CAG-3′)31 condensates at 50 mM and 30 mM MgCl2 as a function of (5′-CAG-3′)31 concentration: population of condensed RNA (g), T2 relaxation constant of condensed RNA (h) and direct comparison of the population of condensed RNA with the resulting R2,water (i). j, Scheme of (5′-CAG-3′)31 partitioning as a function of MgCl2 concentration. Partition coefficient of (5′-CAG-3′)31 is indicated by the ratio of dilute-phase RNA and the number of schematic RNA condensates. Water molecules and Mg2+ ions are schematically indicated by blue and green circles. b,g–i, All errors indicate parameter uncertainties, which were estimated from the diagonal elements of the variance–covariance matrix resulting from fitting experimental data to the CONDENSE-MT model. Error bars are centred around the resulting parameter value after computational optimization ± parameter uncertainty.
