Abstract
RNA-driven condensation plays a central role in organizing and regulating ribonucleoprotein granules within cells. Disruptions to this process—such as the aberrant aggregation of repeat-expanded RNA—are associated with numerous neurological disorders. Here we study the role of biomolecular condensates in irreversible RNA aggregation. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multi-phasic condensate structures, with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of RNA clustering is determined by sequence, secondary structure and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA–RNA interactions and prevents RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation and highlights the chaperone-like function of RNA-binding proteins against aberrant RNA phase transitions.
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Data availability
All data are available in the Article or its Supplementary Information. Source data are provided with this paper. The source microscopy data are available via Dryad at https://doi.org/10.5061/dryad.cc2fqz6hn (ref. 90).
Code availability
Codes for nanorheology, SAC, RNA state diagram, co-localization analyses, condensate fusion assay and complex shear moduli estimation are available via GitHub at https://github.com/BanerjeeLab-repertoire/Biomolecular-Condensates-Can-Enhance-Pathological-RNA-Clustering (ref. 86).
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Acknowledgements
This work was supported by the US National Institutes of Health through grants R35 GM138186 (P.R.B.) and the St. Jude Children’s Research Collaborative on the Biology and Biophysics of RNP Granules (P.R.B.). The funders had no role in the study design, data collection and analysis, the decision to publish or the preparation of the manuscript. We gratefully acknowledge P. Taylor’s laboratory at St. Jude Children’s Research Hospital for providing purified G3BP1 protein. We also gratefully acknowledge I. Alshareedah, currently at Boston Children’s Hospital, Harvard Medical School, for providing the initial SAC analysis codes. We deeply appreciate critical feedback from R. Pappu and T. Mittag and the group members of Banerjee laboratory.
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Conceptualization: P.R.B. and T.S.M.; methodology: P.R.B., T.S.M., A.S. and G.M.W.; investigation: P.R.B., T.S.M., A.S. and G.M.W. Specifically, T.S.M. performed all confocal imaging experiments and data analysis; A.S. performed VPT-based nanorheology measurements and SAC analysis; G.M.W. performed temperature-controlled microscopy of RNA samples; R.G. participated only in the development phase of this study. P.R.B. supervised all experiments and data analysis. Resources: P.R.B.; writing—original draft and revisions: P.R.B., G.M.W. and T.S.M.; writing—reviewing and editing: P.R.B., T.S.M., A.S. and G.M.W.; funding acquisition: P.R.B.
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P.R.B. is a member of the Biophysics Reviews (AIP Publishing) editorial board. This affiliation did not influence the work reported here. All other authors declare no conflicts of interest.
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Extended data
Extended Data Fig. 1 TERRA clustering propensity is independent of the bulk RNA concentration.
Effect of titration of bulk RNA concentration in (a) (TERRA)10 and (b) (TERRA)4 containing ternary RGG-d(T)40 condensate systems on RNA cluster formation upon aging. The condensates and RNA clusters were visualized using FAM-labeled (TERRA)4. The composition of the condensate systems used here is 5 mg/ml RGG, variable d(T)40, and variable (TERRA)10 concentrations (as indicated in the figure panels), with the total nucleic acid concentration kept constant at 2.5 mg/ml. TERRA concentration (in mg/ml and molarity) used in each sample is indicated in each panel of the figure. The buffer contained 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration of FAM labeled (TERRA)4 is 250 nM. Each measurement was independently repeated three times.
Extended Data Fig. 2 G-tracts determine the clustering propensity of TERRA independent of the G-quadruplex structure.
(a) Fluorescence images of WT (TERRA)10 and (mut6GtoU-TERRA)10 containing RGG-d(T)40 ternary condensates at different time points after sample preparation. (b) Reports Thioflavin T (ThT) fluorescence images of (mut6GtoU-TERRA)10 containing RGG-d(T)40 condensates at the indicated time points along with line profiles (shown in green) and corresponding ThT intensity profiles. The ThT intensity profiles for RGG-(TERRA)10-d(T)40 ternary condensates are shown (grey; data is taken from Fig. 2h) for comparison purposes. The composition of the condensate system used here is 1 mg/ml RNA [(TERRA)10, 50.7 μΜ; (mut6GtoU-TERRA)10, 51.3 μΜ], 5 mg/ml RGG, and 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. RNA clusters (or a lack thereof) were probed using 250 nM to 500 nM SYTO-13 in (a). The concentration of ThT used in (b) is 50 μΜ. These experiments were independently repeated three times.
Extended Data Fig. 3 Intra-condensate demixing of RNA clusters in a time-dependent manner.
(a) The white dashed lines shown here correspond to line profile analyses shown in (b) and (c). Pairwise line profile analyses of (TERRA)4 images with respect to RGG (b) and d(T)40 (c) as a function of time. Each line profile shown here is normalized with respect to the maximum intensity value, wherein all values were first offset by the minimum intensity value. The composition of the condensate system used here is 1 mg/ml (TERRA)4 (corresponds to 127 μΜ), 5 mg/ml RGG, and 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration range of labeled components is 250 nM. Each measurement was independently repeated three times.
Extended Data Fig. 4 GC-rich repeat expanded RNAs form intra-condensate clusters in a length-dependent manner.
Fluorescence images utilizing SYTO-13 and Cy5-d(T)40 of RGG-d(T)40 condensates containing either r(CAG) or r(CUG) repeat RNAs corresponding to data shown in Fig. 4b–e. The composition of the condensate system used here is 1 mg/ml RNA [0.45 mg/ml in the case of r(CUG)47; r(CAG)20, 51.2 μM; r(CAG)31, 33 μM; r(CUG)31, 33.8 μM; r(CUG)47, 10 μM], 5 mg/ml RGG, and 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration range of labeled components is 250 nM to 500 nM. Each measurement was independently repeated three times.
Extended Data Fig. 5 Fluorescence recovery after photobleaching (FRAP) reveals dynamical arrest of the RNA component, TERRA, but not RGG and d(T)40 with condensate aging.
(a) Schematic of FRAP assay in condensate. (b) Fluorescence images of (TERRA)4 containing RGG-d(T)40 condensates at pre-bleach, bleach, and post-bleach steps of FRAP experiments corresponding to Fig. 5c and Supplementary Videos 11–16. Shaded regions in each plot signify the standard error. The composition of the (TERRA)4 containing RGG-d(T)40 condensate system used here is 1 mg/ml (TERRA)4 (corresponds to 127 μM), 5 mg/ml RGG, and 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration of labeled components is 250 nM. Each experiment was independently repeated three times.
Extended Data Fig. 6 Cluster size measurements in quaternary condensate systems show the efficacy of ASO or G3BP1 in buffering intra-condensate RNA clustering.
The cluster size plots from SAC at distinct sample ages for (a) ASO-treated (TERRA)10 containing ternary condensate system, (b) G3BP1-treated (TERRA)10 containing ternary condensate system, (c) G3BP1-treated r(CAG)31 containing ternary condensate system, and (d) G3BP1-treated r(CUG)47 containing ternary condensate system. The detection limit of SAC is demarcated (see Methods for further details). All box plot elements are defined similarly to Fig. 2d. The concentrations of the ASO and G3BP1 are 1 mg/ml and 10 μM, respectively. The composition of the ternary condensate system used in these experiments is 1 mg/ml RNA [0.45 mg/ml in the case of r(CUG)47; (TERRA)10, 101 μΜ, r(CAG)31, 33 μM; r(CUG)47, 10 μM], 5 mg/ml RGG, and 1.5 mg/ml d(T)40. Buffer composition for all experiments is 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration range of labeled components is 250 nM to 500 nM. The sample size for each condition is 10 condensates, representative of three biological replicates.
Extended Data Fig. 7 Scrambled ASO is unable to obstruct intra-condensate RNA clustering.
Fluorescence images utilizing FAM-(TERRA)4 and Cy5-d(T)40 of (TERRA)10 containing RGG-d(T)40 condensates treated with a scrambled version of TERRA antisense oligonucleotide (ASO): CACUAC. The composition of the condensates is 1.0 mg/ml (TERRA)10 (corresponds to 50.7 μM), 5.0 mg/ml RGG, 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, 20 mM DTT. 1.0 mg/ml ASO was added to the buffer prior to condensate formation. The concentration range of labeled components is 250 nM to 500 nM. This experiment was independently repeated three times.
Extended Data Fig. 8 G3BP1 buffers intra-condensate RNA clustering.
(top) Fluorescence images utilizing FAM-(TERRA)4 and Cy5-d(T)40 of RGG-d(T)40 condensates containing (TERRA)10 along with G3BP1. (bottom) Cluster sizes derived from SAC are reported. The detection limit of SAC is demarcated. These observations correspond to data shown in Fig. 6. The composition of the condensate system used here is 10 μM G3BP1, 1 mg/ml RNA (corresponds to 50.7 μM), 5 mg/ml RGG, and 1.5 mg/ml d(T)40 in a buffer containing 25 mM Tris-HCl (pH 7.5), 25 mM NaCl, and 20 mM DTT. The concentration of labeled components is 250 nM. The sample size for each condition is 10 condensates, representative of three biological replicates.
Extended Data Fig. 9 A schematic showing the proposed model of intra-condensate RNA percolation and heterotypic buffering.
(a) A model of multi-component condensates formed by two RNAs with strong (as shown in green) and weak (as shown in orange) percolation propensity, respectively, and an RBP. (b) Three possible scenarios of RNA percolation-driven condensate aging or a lack thereof in the presence of a multivalent RBP. (c) Zoomed-in views of the panels shown above.
Supplementary information
Supplementary Information
Supplementary Methods, Figs. 1–35, Notes 1–7, Tables 1 and 2, video legends and references.
Supplementary Video 1
Time-lapse fluorescence imaging showing cluster formation in RGG–d(T)40 condensates containing (TERRA)10, as visualized by SYTO-13 fluorescence. The composition of the condensate system used here is 1 mg ml−1 (TERRA)10, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 500 nM. Scale bar, 10 μm.
Supplementary Video 2
Addition of 1.5 mg ml−1 d(T)40, doped with Cy5-labelled d(T)40, to RGG–(TERRA)10 condensates visualized with FAM-(TERRA)4 fluorescence. The composition of the binary condensate system used here is 1 mg ml−1 (TERRA)10 and 5 mg ml−1 RGG in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 3
Temperature-controlled microscopy showing (TERRA)10 phase separation coupled to percolation; 1 mg ml−1 (TERRA)10 in a buffer composed of 50 mM HEPES (pH 7.5), 6.25 mM Mg2+.
Supplementary Video 4
Temperature-controlled microscopy showing shape relaxation and persistence of percolated (TERRA)10 condensates; 1 mg ml−1 (TERRA)10 in buffer composed of 50 mM HEPES (pH 7.5), 10 mM Mg2+.
Supplementary Video 5
Temperature-controlled microscopy showing reversible phase separation of (mut-TERRA)10; 1 mg ml−1 (mut-TERRA)10 in buffer composed of 50 mM HEPES pH 7.5, 25 mM Mg2+.
Supplementary Video 6
Active fusion of (TERRA)10-containing RGG–d(T)40 condensates at 20 min of age visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 1 mg ml−1 (TERRA)10, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 7
Active fusion of (TERRA)10-containing RGG–d(T)40 condensates at 150 min of age visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 1 mg ml−1 (TERRA)10, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 8
Dissolution of the condensate shell but not the inner RNA clusters using 455 mM NaCl in a 6-h-aged RGG–d(T)40 condensate containing (TERRA)10, visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 0.5 mg ml−1 (TERRA)10, 2.5 mg ml−1 RGG and 0.75 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 9
Dissolution of the condensate shell but not the inner RNA clusters using 455 mM NaCl in a 24-h-aged RGG–d(T)40 condensate containing (TERRA)10, visualized by FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 1 mg ml−1 (TERRA)10, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 10
Dissolution of freshly prepared (15 min of age) RGG–d(T)40 condensates containing (TERRA)10 using 455 mM NaCl, visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 0.5 mg ml−1 (TERRA)10, 2.5 mg ml−1 RGG and 0.75 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 11
FRAP of FAM-(TERRA)4 at the 0-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 12
FRAP of A594-RGG at the 0-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 13
FRAP of Cy5-d(T)40 at the 0-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 14
FRAP of FAM-(TERRA)4 at the 8-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 15
FRAP of A594-RGG at the 8-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 16
FRAP of Cy5-d(T)40 at the 8-h timepoint in (TERRA)4-containing RGG–d(T)40 condensates. Scale bar, 10 μm. The composition of the condensate system used here is 1 mg ml−1 (TERRA)4, 5 mg ml−1 RGG and 1.5 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled component is 250 nM.
Supplementary Video 17
VPT of 200-nm beads inside (TERRA)10-containing RGG–d(T)40 condensates at the 15-min timepoint after sample preparation. The composition of the condensate system used here is 2 mg ml−1 (TERRA)10, 10 mg ml−1 RGG and 3 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT.
Supplementary Video 18
VPT of 200-nm beads inside (TERRA)10-containing RGG–d(T)40 condensates at the 45-min timepoint after sample preparation. The composition of the condensate system used here is 2 mg ml−1 (TERRA)10, 10 mg ml−1 RGG and 3 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT.
Supplementary Video 19
VPT of 200-nm beads inside (TERRA)10-containing RGG–d(T)40 condensates at the 150-min timepoint after sample preparation. The composition of the condensate system used here is 2 mg ml−1 (TERRA)10, 10 mg ml−1 RGG and 3 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT.
Supplementary Video 20
Addition of 1 mg ml−1 ASO (r(CCCUAA)) to 5-h-aged RGG–d(T)40 condensates with RNA clusters of (TERRA)10, visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 0.5 mg ml−1 (TERRA)10, 2.5 mg ml−1 RGG and 0.75 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Video 21
Addition of 1 mg ml−1 ASO (r(CCCUAA)) to 18-h-aged RGG–d(T)40 condensates with RNA clusters of (TERRA)10, visualized with FAM-(TERRA)4 and Cy5-d(T)40 fluorescence. The composition of the condensate system used here is 0.5 mg ml−1 (TERRA)10, 2.5 mg ml−1 RGG and 0.75 mg ml−1 d(T)40 in a buffer containing 25 mM Tris–HCl (pH 7.5), 25 mM NaCl and 20 mM DTT. The concentration of the labelled components is 250 nM.
Supplementary Data 1
Source data for Supplementary Figs. 6, 7, 9, 10, 12–14, 17, 19–22, 25, 28, 31, 33 and 34, for example, statistical source data.
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Source Data Figs. 1–6
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Source Data Extended Data Figs. 2, 3, 6 and 8
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Mahendran, T.S., Wadsworth, G.M., Singh, A. et al. Homotypic RNA clustering accompanies a liquid-to-solid transition inside the core of multi-component biomolecular condensates. Nat. Chem. 17, 1236–1246 (2025). https://doi.org/10.1038/s41557-025-01847-3
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DOI: https://doi.org/10.1038/s41557-025-01847-3