Extended Data Figure 1. Disease-associated repeat-containing RNAs form clusters in vitro.

(a) Fluorescence micrographs for RNA with various GC-content compared against RNA with disease-associated repeat expansions. Sequences of the DNA templates used for transcription are provided in Supplementary Table 2. (b) Fluorescence micrographs comparing 47xCUG RNA and a corresponding control RNA (Scr1) with identical base composition but with a scrambled sequence. Similarly, 47xCAG RNA was compared with two different RNAs (Scr2, Scr3) that have same base composition as 47xCAG but the sequences are scrambled. Extent of inhomogeneity is quantified by the index of dispersion (σ²/μ) across >20 independent imaging areas (1800 μm² each). Each datum point represents an independent imaging area. (c) Representative micrographs of 47xCAG RNA clusters at indicated concentrations. Spherical RNA clusters are observable up to 25 nM RNA concentration. Below this concentration regime, the reaction is reactant limited and the cluster size is below the diffraction limit. RNA clustering at all concentrations was not observed in the presence of 100 mM ammonium acetate. Representative images at indicated RNA concentration in the presence of 100 mM NH₄OAc (+ NH₄OAc). (d) RNA enrichment in the clusters. (Left) Cy3-labeled 66xCAG RNA was serially diluted in conditions that prevent RNA clustering (10 mM Tris pH 7.0, 10 mM MgCl₂, 100 mM NaCl), and the bulk solution fluorescence was calibrated against the RNA concentration. The enrichment of RNA in the clusters was determined by comparing their fluorescence intensity against this calibration. When the input RNA concentration is 100 ng/μl, the concentration in the clusters corresponds to ~16.3 μg/μl, or an enrichment of 163-fold. (Right) RNA clusters were precipitated by centrifugation at 16,000xg for 10 min at room temperature. The concentration of the soluble RNA after centrifugation was determined by measuring absorbance at 260 nm. The concentration of the RNA in the solution phase decreases with the increasing CAG-repeat number. (e, f) 47xCAG RNA clusters were treated with proteinase K (60 U/ml), DNaseI (200 U/ml) or RNaseA (0.7 U/ml) for 10 min at room temperature. Representative micrographs (e) and quantification (f). (g) 47xCAG RNA clustering is inhibited by NaCl. (h) Binary phase diagram for 1.25 μM 47xCAG RNA as a function of MgCl₂ and NaCl concentrations. Blue dots represent two-phase regime while the red dots indicate a homogenous single-phase regime. (i) The RNA clusters are in a solid-like state and do not exhibit fluorescence recovery upon photobleaching, as indicated by the representative micrographs for 47xCUG (top) and 47xCAG (bottom) RNA at the indicated time-points. (j) Sample images showing aborted fusion events between 47xCAG RNA clusters suggesting that the clusters were liquid-like initially and later undergo a liquid-to-solid transition. Fusion events are likely aborted as the clusters solidified prior to relaxation to a spherical geometry. Scale bars in (a, b, c, e, g) are 5 μm and in (i, j) are 1 μm. Error bars depict median and interquartile range. Data are representative of ≥ 3 independent experiments across ≥ 2 independent RNA preparations.