DOI: 10.5061/dryad.cc2fqz6hn
title: Homotypic RNA clustering accompanies a liquid-to-solid transition inside the core of multi-component biomolecular condensates

description: 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 report that irreversible RNA
 aggregation is facilitated within multicomponent protein–nucleic acid
 condensates. Using time-lapse confocal fluorescence microscopy, we tracked
 the growth of RNA clusters as a function of condensate age and found that
 RNA aggregation is driven by RNA percolation, proceeding via a transition
 from intramolecular to intermolecular RNA–RNA interactions. For GC-rich
 repeat RNAs, the size of RNA clusters increases, and the timescale of
 cluster formation decreases with the number of repeat units. Utilizing
 nanorheology, optical tweezer-based droplet fusion assays, and
 high-resolution confocal fluorescence imaging, we uncovered that RNA
 clustering drives a liquid-to-solid phase transition in the condensate
 core, leading to the emergence of multiphasic condensate structures.
 Multivalent RNA-binding cofactors, such as antisense oligonucleotides and
 RNA-binding proteins, that compete with homotypic RNA–RNA interactions can
 raise the activation energy barrier for RNA clustering, thereby acting as
 inhibitors of intra-condensate RNA aggregation. The insights gained from
 this study provide a complementary perspective on the role of
 multicomponent biomolecular condensates in regulating aberrant RNA
 self-assembly in living cells.
description: # Homotypic RNA clustering accompanies a liquid-to-solid transition inside
 the core of multi-component biomolecular condensates This readme file was
 generated on 2025-07-15 by Tharun Selvam Mahendran ## DESCRIPTION: The
 dataset provided here contains imaging data (figures/videos) corresponding
 to the main text, extended data, and supplementary information concerning
 the referenced publication. Methodological details and additional
 acquisition parameters can be found in the relevant methods section of the
 publication. The images are organized into folders, broadly categorized
 into main text figures (Dryad_source_data_for_main_text.zip), extended
 data figures (Dryad_source_data_for_extended_data.zip), and supplementary
 information figures/videos
 (Dryad_source_data_for_supplementary_information.zip). The main folders
 include subfolders specified for individual image panels if the concerned
 figure includes multiple panels of data. These subfolders are named
 following the corresponding panel letter, e.g., a, b, c, d, etc. Further,
 the series of images constituting a figure are categorized according to
 experimental parameters if multiple image data exists in a single figure
 panel: timepoint of imaging (mentioned in hours, minutes or seconds),
 fluorophore being imaged (e.g., SYTO13, Alexa594 or A594, Cy5, FAM, ThT),
 and sample composition (e.g., RGG peptide, dT40 ssDNA, TERRA RNA, CAG31
 repeat RNA), etc. A description is provided along with each set of files
 to provide clarification on technical terms used. Additionally, a direct
 correspondence exists between the organized data in the repository and the
 data shown in the published article. Therefore, details of the sample and
 additional information about the sample conditions, if applicable, can be
 found in the publication, specifically, the relevant figure legend and/or
 method section. ## GENERAL INFORMATION: Title of Dataset: Homotypic RNA
 clustering accompanies a liquid-to-solid transition inside the core of
 multi-component biomolecular condensates Author/Principal Investigator
 Information Name: Priya R. Banerjee ORCID:
 [https://orcid.org/0000-0002-6169-1461](https://orcid.org/0000-0002-6169-1461) Institution: The State University of New York at Buffalo Address: 229 Fronczak Hall Email: [prbanerj@buffalo.edu](mailto:prbanerj@buffalo.edu) Author Name: Tharun Selvam Mahendran ORCID: [https://orcid.org/0000-0002-7536-1236](https://orcid.org/0000-0002-7536-1236) Institution: The State University of New York at Buffalo Address: 206 Fronczak Hall Email: [tharunse@buffalo.edu](mailto:tharunse@buffalo.edu) Author/Alternate Contact Information Name: Anurag Singh ORCID: [https://orcid.org/0000-0002-8132-903X](https://orcid.org/0000-0002-8132-903X) Institution: The State University of New York at Buffalo Address: Email: [singh26@buffalo.edu](mailto:singh26@buffalo.edu) Author/Alternate Contact Information Name: Gable M Wadsworth ORCID: [https://orcid.org/0000-0002-0417-9613](https://orcid.org/0000-0002-0417-9613) Institution: The State University of New York at Buffalo Address: Email: [gablewad@buffalo.edu](mailto:gablewad@buffalo.edu) Date of data collection: between 2023-02-01 and 2025-01-20 Geographic location of data collection: Buffalo, New York, United States of America Information about funding sources that supported the collection of the data: 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.). ## SHARING/ACCESS INFORMATION Licenses/restrictions placed on the data: CC Zero Links to publications that cite or use the data: [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) Links to other publicly accessible locations of the data: Links/relationships to ancillary data sets: [https://github.com/BanerjeeLab-](https://github.com/BanerjeeLab-) repertoire/Biomolecular-Condensates-Can-Enhance-Pathological-RNA-Clustering Was data derived from another source? NO If yes, list source(s): Recommended citation for this dataset: 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. (2025). [https://doi.org/10.1038/s41557-025-01847-3](https://doi.org/10.1038/s41557-025-01847-3) Usage notes: File types are either in .tif or .png formats. Either file type can be accessed via Fiji/ImageJ, a freely available software. ## DATA & FILE OVERVIEW File List: ### &gt;Source data for the main text (contains all the microscopy data shown in the main text)  Dryad_source_data_for_main_text.zip #### Figure 1 * c * 0 hr.tif * 24 hr.tif Description: These image files correspond to different time points. hr indicates hours. * d * Merge.tif * Cy5 dT40.tif * A594 RGG.tif * SYTO-13.tif Description: These image files correspond to different fluorescence channels, e.g., Cy5 dT40 refers to fluorescence channel corresponding to Cy5 dT40 excitation/emission. * e * 1.5 hr.tif * 2 hr.tif * 5 hr.tif * 5.25 hr.tif * 5.5 hr.tif Description: These image files correspond to different time points. hr indicates hours. * f * Merge.tif * A594-RGG.tif * SYTO-13.tif Description: These image files correspond to different fluorescence channels, e.g., A594-RGG refers to fluorescence channel corresponding to A594-RGG excitation/emission. * h * Merge.tif * Cy5-dT40.tif * SYTO-13.tif Description: These image files correspond to different fluorescence channels, e.g., Cy5-dT40 refers to fluorescence channel corresponding to Cy5 dT40 excitation/emission. * j * z stacks.tif Description: Individual z slices in the form of an image stack. #### Figure 2 * b * 20C.tif * 90C.tif * Cooled to 20C.tif Description: 'C' here refers to degrees Celsius, e.g., 20C refers to 20 degrees Celsius. * d * 1st from left.tif * 2nd from left.tif * 3rd from left.tif * 4th from left.tif * 5th from left.tif * 6th from left.tif * 7th from left.tif * 8th from left.tif Description: The naming order described here is in reference to a series of images, corresponding to imaging data from measurements involving temperature ramps. The '1st from left.tif' is the start of the order and '8th from left.tif' is the end. * e * 20C.tif * 40C.tif * Cooled to 20C.tif Description: 'C' here refers to degrees Celsius, e.g., 20C refers to 20 degrees Celsius. * g * 0 hr SYTO-13.tif * 0 hr A594-RGG.tif * 0 hr Merge.tif * 24 hr SYTO-13.tif * 24 hr A594-RGG.tif * 24 hr Merge.tif Description: These image files correspond to distinct time points and fluorescence channels. For example, 0 hr SYTO-13 refers to the 0-hour time point with SYT0-13 fluorescence channel. * h * TERRAx10 0hr.tif * TERRAx10 24hr.tif * mut-TERRAx10 0hr.tif * mut-TERRAx10 24hr.tif Description: These image files correspond to the RNA used in sample preparation and the time point. For example, TERRAx10 0hr refers to the RNA, TERRAx10, and the time point of 0 hours; 'x10' in TERRAx10 refers to number of RNA repeat units. #### Figure 3 * a * TERRAx1 0hr.tif * TERRAx1 2hr.tif * TERRAx1 4hr.tif * TERRAx1 8hr.tif * TERRAx1 18hr.tif * TERRAx4 0hr.tif * TERRAx4 2hr.tif * TERRAx4 4hr.tif * TERRAx4 8hr.tif * TERRAx4 18hr.tif * TERRAx6 0hr.tif * TERRAx6 2hr.tif * TERRAx6 4hr.tif * TERRAx6 8hr.tif * TERRAx6 18hr.tif * TERRAx10 0hr.tif * TERRAx10 2hr.tif * TERRAx10 4hr.tif * TERRAx10 8hr.tif * TERRAx10 18hr.tif Description: These image files correspond to the RNA used in sample preparation and the time point. For example, TERRAx10 0hr refers to the RNA, TERRAx10, and the time point of 0 hours; 'x10' in TERRAx10 refers to the number of RNA repeat units. * b * Image 0 hr * spatial autocorrelation 0 hr * Image 18 hr * spatial autocorrelation 18 hr * Description: These files are categorized either as the image files or the corresponding spatial autocorrelation plots with the time point indicated wherein hr means hours. #### Figure 4 * b * CAGx20 SYTO-13 0hr.tif * CAGx20 A594 RGG 0hr.tif * CAGx20 Merge 0hr.tif * CAGx20 SYTO-13 24hr.tif * CAGx20 A594 RGG 24hr.tif * CAGx20 Merge 24hr.tif * CAGx31 SYTO-13 0hr.tif * CAGx31 A594 RGG 0hr.tif * CAGx31 Merge 0hr.tif * CAGx31 SYTO-13 24hr.tif * CAGx31 A594 RGG 24hr.tif * CAGx31 Merge 24hr.tif Description: These image files correspond to the RNA used in sample preparation, the fluorescence channel, and the time point. For example, CAGx31 SYTO-13 0hr refers to the RNA, CAGx31; the fluorescence channel, SYTO-13; the time point of 0 hours; 'x31' in CAGx31 refers to the number of RNA repeat units. * d * CUGx31 SYTO-13 0hr.tif * CUGx31 A594 RGG 0hr.tif * CUGx31 Merge 0hr.tif * CUGx31 SYTO-13 24hr.tif * CUGx31 A594 RGG 24hr.tif * CUGx31 Merge 24hr.tif * CUGx47 SYTO-13 0hr.tif * CUGx47 A594 RGG 0hr.tif * CUGx47 Merge 0hr.tif * CUGx47 SYTO-13 24hr.tif * CUGx47 A594 RGG 24hr.tif * CUGx47 Merge 24hr.tif Description: These image files correspond to the RNA used in sample preparation, the fluorescence channel, and the time point. For example, CAGx31 SYTO-13 0hr refers to the RNA, CAGx31; the fluorescence channel, SYTO-13; the time point of 0 hours; 'x31' in CAGx31 refers to the number of RNA repeat units. #### Figure 5 * a * 20 min before fusion.tif * 20 min after fusion.tif * 150 min before fusion.tif * 150 min after fusion.tif Description: Here, the files are organized in terms of time point and the condition of 'after fusion' and 'before fusion'. For example, 20 min before fusion refers to 20 minute time point, and the image collected before fusion event * b * Before.tif * 6 s.tif * 8 s.tif * 10 s.tif * 16 s.tif Description: Here, the files are organized according to time point wherein s refers to seconds. 'Before' refers to the initial time point or 0 s data. For example, 8 s refers to 8 seconds. #### Figure 6 * b * FAM TERRAx4 0hr.tif * A594 RGG 0hr.tif * Merge 0 hr.tif * FAM TERRAx4 24hr.tif * A594 RGG 24hr.tif * Merge 24 hr.tif Description: These image files correspond to the fluorescence channel, and the time point. For example, FAM TERRAx4 0hr refers to the fluorescence channel, FAM TERRAx4; the time point of 0 hours; 'x4' in FAM TERRAx4 refers to the number of RNA repeat units. * d * before.tif * 1 hr.tif * 2.25 hr.tif Description: Here, the files are organized according to time point wherein hr refers to hours. 'Before' refers to the initial time point or 0 hr data. For example, 1 hr refers to 1 hour. * f * FAM TERRAx4 0hr.tif * A594 RGG 0hr.tif * Merge 0 hr.tif * FAM TERRAx4 24hr.tif * A594 RGG 24hr.tif * Merge 24 hr.tif Description: These image files correspond to the fluorescence channel, and the time point. For example, FAM TERRAx4 0hr refers to the fluorescence channel, FAM TERRAx4; the time point of 0 hours; 'x4' in FAM TERRAx4 refers to the number of RNA repeat units. * i * SYTO13 0hr.tif * A594 RGG 0hr.tif * Merge 0 hr.tif * SYTO13 24hr.tif * A594 RGG 24hr.tif * Merge 24 hr.tif Description: These image files correspond to the fluorescence channel, and the time point. For example, SYTO13 0hr refers to the fluorescence channel, SYTO13; the time point of 0 hours. * j * SYTO13 0hr.tif * A594 RGG 0hr.tif * Merge 0 hr.tif * SYTO13 24hr.tif * A594 RGG 24hr.tif * Merge 24 hr.tif Description: These image files correspond to the fluorescence channel, and the time point. For example, SYTO13 0hr refers to the fluorescence channel, SYTO13; the time point of 0 hours. \&gt;Source data for supplementary text (contains all the microscopy data shown in the supplementary text including videos) ### Dryad_source_data_for_supplementary_information.zip \&gt;Supplementary Figures #### Figure 1 * 24 hr.tif * 0 hr.tif Description: The files correspond to images collected at different time points. For example, 0 hr refers to 0 hours. #### Figure 2 * 24 hr.tif * 0 hr.tif Description: The files correspond to images collected at different time points. For example, 0 hr refers to 0 hours. #### Figure 3 * SYTO-13 0hr.tif * A594-RGG 0hr.tif * Merge 0 hr.tif * SYTO-13 24hr.tif * A594-RGG 24hr.tif * Merge 24 hr.tif Description: These image files correspond to the fluorescence channel, and the time point. For example, SYTO-13 0hr refers to the fluorescence channel, SYTO-13; the time point of 0 hours. #### Figure 4 * Image sequence of timelapse.tif Description: A sequence of fluorescence images constituting a timelapse from beginning to end. #### Figure 5 * 5%.tif * 10%.tif * 20%.tif Description: Here the files are organized in terms of the crowder percentage. #### Figure 6 * a * 0 ugml.tif * 8 ugml.tif * 78 ugml.tif * 196 ugml.tif * 314 ugml.tif * 444 ugml.tif Description: Here, the files are organized in terms of the RNA concentration expressed as ugml, which means micrograms/milliliters. * e * 0 hr.tif * 24 hr.tif Description: Here, the files are organized in terms of time points. #### Figure 8 * 0 hr SYTO-13.tif * 0 hr Cy5-dT40.tif * 0 hr Merge.tif * 24 hr SYTO-13.tif * 24 hr Cy5-dT40.tif * 24 hr Merge.tif Description: These files are organized in terms of time point and fluorescence channel. For example, 0 hr SYTO-13 refers to 0 hour timepoint and SYTO-13 fluorescence channel. #### Figure 10 * RGG dT40 condensates 0 hr.tif * RGG dT40 condensates 24 hr.tif * RGG dT40 polyU condensates 0 hr.tif * RGG dT40 polyU condensates 24 hr.tif Description: These image files are organized according to the condensate sample, and the time point. For example, RGG dT40 condensates 0 hr refers to the RGG dT40 condensates and the time point of 0 hours. #### Figure 11 * NaCl treated condensates SYTO-13 channel 0 hr.tif * NaCl treated condensates Cy5-dT40 channel 0 hr.tif * NaCl treated condensates Merge 0 hr.tif * NaCl treated condensates SYTO-13 channel 2.5 hr.tif * NaCl treated condensates Cy5-dT40 channel 2.5 hr.tif * NaCl treated condensates Merge 2.5 hr.tif * NaCl treated condensates SYTO-13 channel 24 hr.tif * NaCl treated condensates Cy5-dT40 channel 24 hr.tif * NaCl treated condensates Merge 24 hr.tif * KCl treated condensates SYTO-13 channel 0 hr.tif * KCl treated condensates Cy5-dT40 channel 0 hr.tif * KCl treated condensates Merge 0 hr.tif * KCl treated condensates SYTO-13 channel 2.5 hr.tif * KCl treated condensates Cy5-dT40 channel 2.5 hr.tif * KCl treated condensates Merge 2.5 hr.tif * KCl treated condensates SYTO-13 channel 24 hr.tif * KCl treated condensates Cy5-dT40 channel 24 hr.tif * KCl treated condensates Merge 24 hr.tif * LiCl treated condensates SYTO-13 channel 0 hr.tif * LiCl treated condensates Cy5-dT40 channel 0 hr.tif * LiCl treated condensates Merge 0 hr.tif * LiCl treated condensates SYTO-13 channel 2.5 hr.tif * LiCl treated condensates Cy5-dT40 channel 2.5 hr.tif * LiCl treated condensates Merge 2.5 hr.tif * LiCl treated condensates SYTO-13 channel 24 hr.tif * LiCl treated condensates Cy5-dT40 channel 24 hr.tif * LiCl treated condensates Merge 24 hr.tif * NH4OAc treated condensates Cy5-dT40 channel 0 hr.tif * NH4OAc treated condensates Merge 0 hr.tif * NH4OAc treated condensates SYTO-13 channel 2.5 hr.tif * NH4OAc treated condensates Cy5-dT40 channel 2.5 hr.tif * NH4OAc treated condensates Merge 2.5 hr.tif * NH4OAc treated condensates SYTO-13 channel 24 hr.tif * NH4OAc treated condensates Cy5-dT40 channel 24 hr.tif * NH4OAc treated condensates Merge 24 hr.tif Description: The files are organized in terms of chemical treatment, fluorescence channel, and time points. For example, LiCl treated condensates Merge 24 hr refers to a sample with LiCl treatment, the Merge channel, and 24 hours time point. #### Figure 12 * panel A * panel B * panel C * panel D Description: Each file represents an image or plot corresponding to a particular panel: A, B, C, and D. #### Figure 13 * panel a Image final xy.png * panel a Image final xz.png * panel a Image final yz.png * panel b Confocal image - 100nm.png * panel b Heat map - 100nm.png * panel b spatial autocorrelation - 100nm.png * panel c Confocal image - 200nm.png * panel c Heat map - 200nm.png * panel c spatial autocorrelation - 200nm.png * panel d Confocal image - 1um.png * panel d Heat map - 1um.png * panel d spatial autocorrelation - 1um.png Description: Here the files are organized in terms of the panel letter and whether the data corresponds to either an image, heat map, or spatial autocorrelation, as well as the size of the bead, such as 100 nm, 200 nm, and 1 um; nm means nanometers and um means micrometers. #### Figure 14 * panel a FAM-TERRA 0 hr.tif * panel a A594-RGG 0 hr.tif * panel a FAM-TERRA 4 hr.tif * panel a A594-RGG 4 hr.tif * panel a FAM-TERRA 8 hr.tif * panel a A594-RGG 8 hr.tif Description: Here the files are organized in terms of the panel letter, fluorescence channel and the time point. For example, panel a FAM-TERRA 0 hr corresponds to data in panel a, FAM-TERRA channel and 0 hour time point. #### Figure 15 * Panel a 0.1mg/ml CAGx31 SYTO-13.tif * Panel a 0.1mg/ml CAGx31 A594-RGG.tif * Panel a 0.1mg/ml CAGx31 Merge.tif * Panel a 0.5mg/ml CAGx31 SYTO-13.tif * Panel a 0.5mg/ml CAGx31 A594-RGG.tif * Panel a 0.5mg/ml CAGx31 Merge.tif * Panel a 1mg/ml CAGx31 SYTO-13.tif * Panel a 1mg/ml CAGx31 A594-RGG.tif * Panel a 1mg/ml CAGx31 Merge.tif * Panel b 0.1mg/ml CAGx31 SYTO-13.tif * Panel b 0.1mg/ml CAGx31 Cy5-dT40.tif * Panel b 0.1mg/ml CAGx31 Merge.tif * Panel b 0.5mg/ml CAGx31 SYTO-13.tif * Panel b 0.5mg/ml CAGx31 Cy5-dT40.tif * Panel b 0.5mg/ml CAGx31 Merge.tif * Panel b 1mg/ml CAGx31 SYTO-13.tif * Panel b 1mg/ml CAGx31 Cy5-dT40.tif * Panel b 1mg/ml CAGx31 Merge.tif Description: Here the files are organized in terms of the panel letter, RNA concentration, and fluorescence channel. For example, Panel b 0.5mg/ml CAGx31 SYTO-13 corresponds to data in panel b, 0.5 mg/ml (corresponds to milligrams/milliliter) of CAGx31 RNA (x31 refers to 31 RNA repeats) and SYTO-13 fluorescence channel. #### Figure 16 * 0 hr SYTO-13.tif * 0 hr Cy5-dT40.tif * 0 hr Merge.tif * 24 hr SYTO-13.tif * 24 hr Cy5-dT40.tif * 24 hr Merge.tif Description: Here the files are organized in terms of the time point and fluorescence channel. For example, 0 hr SYTO-13 refers to 0 hour time point and SYTO-13 fluorescence channel. #### Figure 17 * CAGx20.tif * CAGx31.tif * GGGGCCx5.tif Description: Here the files are organized according to the RNA used in the sample (CAG or GGGGCC). For example, CAGx20 means CAG RNA and here, x20 refers to the number of RNA repeat units, which is 20 in this example. #### Figure 18 * b * Before.tif * 2 s.tif * 4 s.tif Description: Here the files are organized according to time point. 'Before' refers to the initial time point, and s refers to seconds. * c * Before.tif * 2 s.tif * 4 s.tif Description: Here the files are organized according to time point. 'Before' refers to the initial time point, and s refers to seconds. #### Figure 19 * panel a #### Figure 23 * SYTO-13 0 hr.tif * Cy5-dT40 0 hr.tif * Merge 0 hr.tif * SYTO-13 24 hr.tif * Cy5-dT40 24 hr.tif * Merge 24 hr.tif Description: Here the files are organized in terms of the time point and fluorescence channel. For example, SYTO-13 0 hr refers to SYTO-13 fluorescence channel and 0 hour time point. #### Figure 24 * 0 hr FAM TERRA.tif * 0 hr Cy5 dT40.tif * 0 hr composite.tif * 24 hr FAM TERRA.tif * 24 hr Cy5 dT40.tif * 24 hr composite.tif * 48 hr FAM TERRA.tif * 48 hr Cy5 dT40.tif * 48 hr composite.tif * 72 hr FAM TERRA.tif * 72 hr Cy5 dT40.tif * 72 hr composite.tif * 96 hr FAM TERRA.tif * 96 hr Cy5 dT40.tif * 96 hr composite.tif Description: Here the files are organized in terms of time point (hr means hours) and fluorescence channel (FAM TERRA, Cy5 dT40, composite). #### Figure 26 * before.tif * 3 hr.tif * 9 hr.tif Description: Here the files are organized in terms of time points. Before refers to the initial timepoint and hr refers to hours. #### Figure 27 * A488 G3BP1.tif * Cy5 dT40.tif * Composite.tif Description: Here the files are organized in terms of fluorescence channel, A488 G3BP1, Cy5 dT40, or Composite. #### Figure 28 * panel a * 0 uM * 2.5 uM * 5 uM * 10 uM * 15 uM * 23 uM #### Figure 29 * 0 hr SYTO-13.tif * 0 hr Cy5-dT40.tif * 0 hr Merge.tif * 1 hr SYTO-13.tif * 1 hr Cy5-dT40.tif * 1 hr Merge.tif * 2 hr SYTO-13.tif * 2 hr Cy5-dT40.tif * 2 hr Merge.tif * 4 hr SYTO-13.tif * 4 hr Cy5-dT40.tif * 4 hr Merge.tif Description: Here the files are organized in terms of time point (hr means hours) and fluorescence channel (SYTO-13, Cy5-dT40, Merge). #### Figure 30 * TERRAx10 FAM-TERRA 0 hr.tif * TERRAx10 Cy5-dT40 0 hr.tif * TERRAx10 Merge 0 hr.tif * TERRAx10 FAM-TERRA 24 hr.tif * TERRAx10 Cy5-dT40 24 hr.tif * TERRAx10 Merge 24 hr.tif * CAGx31 FAM-TERRA 0 hr.tif * CAGx31 Cy5-dT40 0 hr.tif * CAGx31 Merge 0 hr.tif * CAGx31 FAM-TERRA 24 hr.tif * CAGx31 Cy5-dT40 24 hr.tif * CAGx31 Merge 24 hr.tif * CUGx47 FAM-TERRA 0 hr.tif * CUGx47 Cy5-dT40 0 hr.tif * CUGx47 Merge 0 hr.tif * CUGx47 FAM-TERRA 24 hr.tif * CUGx47 Cy5-dT40 24 hr.tif * CUGx47 Merge 24 hr.tif Description: Here the files are organized in terms of RNA in sample, fluorescence channel and time point. For example, CUGx47 FAM-TERRA 0 hr refers to CUGx47 RNA (x47 refers to number of repeat units), FAM-TERRA fluorescence channel, and the time point, 0 hours. #### Figure 32 * Before addition.tif * Post addition 4 hr.tif * Post addition 24 hr.tif Description: Here the files are organized in terms of before and after the addition of the reagent, imaged at 4 hours and 24 hours. hr means hours. #### Figure 34 * panel a * 0 hr.tif * 24 hr.tif Description: Here the files are organized in terms of time points. hr means hours. * panel b * 0 hr.tif * 24 hr.tif Description: Here the files are organized in terms of time points. hr means hours. #### Figure 35 * a * b * c ### &gt;Supplementary Videos #### Video 1 * Video Description: A series of images compiled as an image stack #### Video 2 * Video.tif Description: A series of images compiled as an image stack #### Video 3 * Video.tif Description: A series of images compiled as an image stack #### Video 4 * Video Description: A series of images compiled as an image stack #### Video 5 * Video.tif Description: A series of images compiled as an image stack #### Video 6 * video Description: A series of images compiled as an image stack #### Video 7 * video.tif Description: A series of images compiled as an image stack #### Video 8 * Timelapse microscopy Description: A series of images compiled as an image stack #### Video 9 * timelapse movie Description: A series of images compiled as an image stack #### Video 10 * timelapse movie Description: A series of images compiled as an image stack #### Video 11 * FAM-TERRA 0 hr Description: Fluorescence recovery after photobleaching (FRAP) measurements in the FAM- TERRA channel at 0 hour timepoint (hr means hours). #### Video 12 * A594-RGG 0hr FRAP Description: Fluorescence recovery after photobleaching (FRAP) measurements in the A594- RGG channel at 0 hour timepoint (hr means hours). #### Video 13 * Cy5-dT40 0 hr Description: Fluorescence recovery after photobleaching (FRAP) measurements in the Cy5- dT40 channel at 0 hour timepoint (hr means hours). #### Video 14 * FAM-TERRA 8 hr Description: Fluorescence recovery after photobleaching (FRAP) measurements in the FAM- TERRA channel at 8 hour timepoint (hr means hours). #### Video 15 * A594-RGG 8 hr Description: Fluorescence recovery after photobleaching (FRAP) measurements in the A594- RGG channel at 8 hour timepoint (hr means hours). #### Video 16 * Cy5-dT40 8 hr Description: Fluorescence recovery after photobleaching (FRAP) measurements in the Cy5- dT40 channel at 8 hour timepoint (hr means hours). #### Video 17 * VPT movie Description: A series of images compiled as an image stack #### Video 18 * VPT movie Description: A series of images compiled as an image stack #### Video 19 * video Description: A series of images compiled as an image stack #### Video 20 * video.tif Description: A series of images compiled as an image stack #### Video 21 * video.tif Description: A series of images compiled as an image stack ### &gt;Source data for extended data (contains all the microscopy data shown in the extended data) Dryad_source_data_for_extended_data.zip #### Figure 1 * a * 0.5mgml 0 hr.tif * 0.5mgml 24 hr.tif * 0.25mgml 0 hr.tif * 0.25mgml 24 hr.tif * 1mgml 0 hr.tif * 1mgml 24 hr.tif Description: Files are organized in terms of concentration expressed in terms of milligrams/milliliter (corresponds to mgml) and time point (hr means hours). * b * 0.5mg/ml 0 hr.tif * 0.5mg/ml 24 hr.tif * 0.25mg/ml 0 hr.tif * 0.25mg/ml 24 hr.tif * 1mg/ml 0 hr.tif * 1mg/ml 24 hr.tif Description: Files are organized in terms of concentration expressed in terms of milligrams/milliliter (corresponds to mg/ml) and time point (hr means hours). #### Figure 2 * a * mut6GtoU 0 hr.tif * mut6GtoU 1 hr.tif * mut6GtoU 2 hr.tif * mut6GtoU 16 hr.tif * WT 0 hr.tif * WT 1 hr.tif * WT 2 hr.tif * WT 16 hr.tif Description: Here the files are organized according to RNA variant, classified as either wild type (WT) or mutant (mut6GtoU), as well as the time point (hr means hours). * b * 0 hr.tif * 24 hr.tif Description: Here the files are organized in terms of time points. hr means hours. #### Figure 3 * A594-RGG image of FAM-TERRA and A594-RGG panel 0 hr.tif * A594-RGG image of FAM-TERRA and A594-RGG panel 4 hr.tif * A594-RGG image of FAM-TERRA and A594-RGG panel 8 hr.tif * Cy5-dT40 image of FAM-TERRA and Cy5-dT40 panel 0 hr.tif * Cy5-dT40 image of FAM-TERRA and Cy5-dT40 panel 4 hr.tif * Cy5-dT40 image of FAM-TERRA and Cy5-dT40 panel 8 hr.tif * FAM-TERRA image of FAM-TERRA and A594-RGG panel 0 hr.tif * FAM-TERRA image of FAM-TERRA and A594-RGG panel 4 hr.tif * FAM-TERRA image of FAM-TERRA and A594-RGG panel 8 hr.tif * FAM-TERRA image of FAM-TERRA and Cy5-dT40 panel 0 hr.tif * FAM-TERRA image of FAM-TERRA and Cy5-dT40 panel 4 hr.tif * FAM-TERRA image of FAM-TERRA and Cy5-dT40 panel 8 hr.tif * Merge image of FAM-TERRA and A594-RGG panel 0 hr.tif * Merge image of FAM-TERRA and A594-RGG panel 4 hr.tif * Merge image of FAM-TERRA and A594-RGG panel 8 hr.tif * Merge image of FAM-TERRA and Cy5-dT40 panel 0 hr.tif * Merge image of FAM-TERRA and Cy5-dT40 panel 4 hr.tif * Merge image of FAM-TERRA and Cy5-dT40 panel 8 hr.tif Description: Here the files are organzied in terms of a fluorescence channel in a pairwise channel combination as well as the time point. For example, A594-RGG image of FAM-TERRA and A594-RGG panel 0 hr refers to A594-RGG fluorescence channel in a pairwise channel combination of FAM-TERRA and A594-RGG, at the 0 hour time point (hr means hours). #### Figure 4 * CAGx20 Cy5-dT40 0 hr.tif * CAGx20 Cy5-dT40 24 hr.tif * CAGx20 Merge 0 hr.tif * CAGx20 Merge 24 hr.tif * CAGx20 SYTO-13 0 hr.tif * CAGx20 SYTO-13 24 hr.tif * CAGx31 Cy5-dT40 0 hr.tif * CAGx31 Cy5-dT40 24 hr.tif * CAGx31 Merge 0 hr.tif * CAGx31 Merge 24 hr.tif * CAGx31 SYTO-13 0 hr.tif * CAGx31 SYTO-13 24 hr.tif * CUGx31 Cy5-dT40 0 hr.tif * CUGx31 Cy5-dT40 24 hr.tif * CUGx31 Merge 0 hr.tif * CUGx31 Merge 24 hr.tif * CUGx31 SYTO-13 0 hr.tif * CUGx31 SYTO-13 24 hr.tif * CUGx47 Cy5-dT40 0 hr.tif * CUGx47 Cy5-dT40 24 hr.tif * CUGx47 Merge 0 hr.tif * CUGx47 Merge 24 hr.tif * CUGx47 SYTO-13 0 hr.tif * CUGx47 SYTO-13 24 hr.tif Description: Here the files are organized in terms of the RNA used in the sample, fluorescence channel and the time point. For example, CAGx20 Cy5-dT40 0 hr refers to a sample containing CAGx20 RNA (x20 refers to 20 RNA repeat units), Cy5-dT40 fluorescence channel and 0 hour time point (hr means hours). #### Figure 5 * A594-RGG 0 hr Bleach.tif * A594-RGG 0 hr Post-bleach.tif * A594-RGG 0 hr Pre-bleach.tif * A594-RGG 8 hr Bleach.tif * A594-RGG 8 hr Post-bleach.tif * A594-RGG 8 hr Pre-bleach.tif * Cy5-dT40 0 hr Bleach.tif * Cy5-dT40 0 hr Post-bleach.tif * Cy5-dT40 0 hr Pre-bleach.tif * Cy5-dT40 8 hr Bleach.tif * Cy5-dT40 8 hr Post-bleach.tif * Cy5-dT40 8 hr Pre-bleach.tif * FAM-TERRA 0 hr Bleach.tif * FAM-TERRA 0 hr Post-bleach.tif * FAM-TERRA 0 hr Pre-bleach.tif * FAM-TERRA 8 hr Bleach.tif * FAM-TERRA 8 hr Post-bleach.tif * FAM-TERRA 8 hr Pre-bleach.tif Description: Here the files are organized in terms of fluorescence channel (A594-RGG, Cy5-dT40, or FAM-TERRA), time point (hr means hours), and assay step (Pre-bleach, Bleach, Post-bleach). #### Figure 7 * 0 hr composite.tif * 0 hr Cy5 dT40.tif * 0 hr FAM TERRA4.tif * 24 hr composite.tif * 24 hr Cy5 dT40.tif * 24 hr FAM TERRA4.tif Description: Here the files are organized in terms of time point (0 hr or 24 hr; hr means hours) and fluorescence channel (FAM TERRA4, Cy5 dT40 or composite). #### Figure 8 * 0 hr composite.tif * 0 hr Cy5 dT40.tif * 0 hr FAM TERRA4.tif * 24 hr composite.tif * 24 hr Cy5 dT40.tif * 24 hr FAM TERRA4.tif * 48 hr composite.tif * 48 hr Cy5 dT40.tif * 48 hr FAM TERRA4.tif * 72 hr composite.tif * 72 hr Cy5 dT40.tif * 72 hr FAM TERRA4.tif * 96 hr composite.tif * 96 hr Cys dT40.tif * 96 hr FAM TERRA4.tif Description: Here the files are organized in terms of the time point (expressed in hours; hr means hours) and fluorescence channel (FAM TERRA4, Cy5 dT40, or composite). Relationship between files, if important: Additional related data collected that was not included in the current data package: Are there multiple versions of the dataset? If yes, name of file(s) that was updated: Why was the file updated? When was the file updated? ## METHODOLOGICAL INFORMATION Description of methods used for collection/generation of data: for methodological details, please see [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) Methods for processing the data: for methodological details, please see [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) Instrument- or software-specific information needed to interpret the data: Files are available in standard formats that are accessible using Fiji. For further details, please see [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) Environmental/experimental conditions: for methodological details, please see [https://www.nature.com/articles/s41557-025-01847-3](https://www.nature.com/articles/s41557-025-01847-3) People involved with sample collection, processing, analysis, and/or submission: Tharun Selvam Mahendran, Gable M Wadsworth, Anurag Singh, Ritika Gupta, and Priya R. BanerjeeThis readme file was generated on 2025-07-15 by Tharun Selvam Mahendran