Undergo Phase Separation Research Articles

A paternal protein facilitates sperm RNA delivery to regulate zygotic development.

Sperm contributes essential paternal factors, including the paternal genome, centrosome, and oocyte-activation signals, to sexual reproduction. However, it remains unresolved how sperm contributes its RNA molecules to regulate early embryonic development. Here, we show that the Caenorhabditis elegans paternal protein SPE-11 assembles into granules during meiotic divisions of spermatogenesis and later matures into a perinuclear structure where sperm RNAs localize. We reconstitute an SPE-11 liquid-phase scaffold in vitro and find that SPE-11 condensates incorporate the nematode RNA, which, in turn, promotes SPE-11 phase separation. Loss of SPE-11 does not affect sperm motility or fertilization but causes pleiotropic development defects in early embryos, and spe-11 mutant males reduce mRNA levels of genes crucial for an oocyte-to-embryo transition or embryonic development. These results reveal that SPE-11 undergoes phase separation and associates with sperm RNAs that are delivered to oocytes during fertilization, providing insights into how a paternal protein regulates early embryonic development.

The C-terminus of WNK1 kinase is evolutionarily conserved and mediates unique phase behavior

With-no-lysine 1 (WNK1) is a serine-threonine kinase with two isoforms, long (L)-WNK1 and kidney-specific (KS)-WNK1. L-WNK1 is an ancient kinase that is activated by hypertonic stress. Alternatively, KS-WNK1 emerged more recently, it is only expressed in the kidney, it is regulated by K+, and it lacks a functional kinase domain. Both WNK1 isoforms contain identical large C-term domains (CTD) with intrinsically disordered regions (IDRs) that drive the protein into discrete puncta. Our research has identified these puncta as membraneless biomolecular condensates that form via phase separation. In cells these condensates are visible at physiologic levels of hypertonic stress and at native levels of protein expression. In the kidney these condensates appear during K+ stress and are termed WNK bodies. We hypothesized that the large CTD of WNK1 mediates its phase behavior and that this function is evolutionarily conserved. To determine whether the CTD is required for condensate formation we performed live cell imaging studies of expressed mRuby2-tagged WNK1 constructs in cells. Full-length L-WNK1 was compared to two truncated constructs (1-494 or 1-1242), revealing that the CTD is required for condensate formation. To screen for the specific regions of the CTD that undergo phase separation, we used an optogenetic approach that mapped two areas within the WNK1 CTD that facilitate phase separation: (480-1242) and (1770-2126). Both regions were intrinsically disordered, exhibited low sequence complexity, and contained coiled-coil and prion-like domains- features typical of proteins that exhibit strong phase behavior. A bioinformatic analysis of human, C. elegan, and Drosophila WNK kinases revealed that these kinases shared nearly identical disorder tendency plots. A deeper analysis of CTD sequence complexity across 27 species ranging from humans to protists revealed that mammalian WNK kinases are enriched in prolines and serines, whereas invertebrates favor glutamine. Thus, the WNK CTD underwent a permissive Q-to-P/S amino acid shift during evolution, likely to preserve phase behavior. This conserved CTD has physiologic implications in the kidney as KS-WNK1 has an identical CTD and undergoes phase separation into WNK bodies during potassium deficiency. Mice that lack KS-WNK1 (KS-WNK1 KO) do not form WNK bodies during potassium deficiency and have a Gitelman-like phenotype with a 33% decrease in phospo-NCC [P = 0.01]. This decrease in pNCC correlated with decreased thiazide sensitivity—KO mice treated with HCTZ (25mg/kg IP) had no significant reduction in blood pressure, whereas WT littermates had a 4.5mmHg reduction in mean arterial pressure [P ≤ 0.05]. Based on these findings we conclude that during evolution KS-WNK1 conserved the intrinsically disordered WNK CTD to assemble WNK bodies to serve as protein scaffolds that enhance NCC activation during K+ stress. R01 DK098145, R01 DK119252, K08 DK118211 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Self-assembly of shell protein and native enzyme in a crowded environment leads to catalytically active phase condensates.

The self-assembly of bacterial microcompartments is the result of several genetic, biochemical, and physical stimuli orchestrating inside the bacterial cell. In this work, we use 1,2-propanediol utilization microcompartments as a paradigm to identify the factors that physically drive the self-assembly of MCP proteins in vitro using its major shell protein and major encapsulated enzyme. We find that a major shell protein PduBB' tends to self-assemble under macromolecular crowded environment and suitable ionic strength. Microscopic visualization and biophysical studies reveal phase separation to be the principle mechanism behind the self-association of shell protein in the presence of salts and macromolecular crowding. The shell protein PduBB' interacts with the enzyme diol-dehydratase PduCDE and co-assemble into phase separated liquid droplets. The co-assembly of PduCDE and PduBB' results in the enhancement of catalytic activity of the enzyme. The shell proteins that make up PduBB' (PduB and PduB') have contrasting self-assembly behavior. While N-terminal truncated PduB' has a high self-associating property and forms solid assemblies that separates out of solution, the longer component of the shell protein PduBM38L is more soluble and shows least tendency to undergo phase separation. A combination of spectroscopic, imaging and biochemical techniques shows the relevance of divalent cation Mg2+ in providing stability to intact PduMCP. Together our results suggest a combination of protein-protein interactions and phase separation guiding the self-assembly of Pdu shell protein and enzyme in the solution phase.

Phase separation of the nuclear pore complex facilitates selective nuclear transport to regulate plant defense against pathogen and pest invasion

The nuclear pore complex (NPC), the sole exchange channel between the nucleus and cytoplasm, is composed of several subcomplexes, among which the central barrier determines the permeability/selectivity of the NPC to dominate the nucleocytoplasmic trafficking essential for many important signaling events in yeast and mammals. How plant NPC central barrier controls selective transport is a crucial question remaining to be elucidated. In this study, we uncovered that phase separation of the central barrier is critical for the permeability and selectivity of plant NPC in the regulation of various biotic stresses. Phenotypic assays of nup62 mutants and complementary lines showed that NUP62 positively regulates plant defense against Botrytis cinerea, one of the world’s most disastrous plant pathogens. Furthermore, in vivo imaging and in vitro biochemical evidence revealed that plant NPC central barrier undergoes phase separation to regulate selective nucleocytoplasmic transport of immune regulators, as exemplified by MPK3, essential for plant resistance to B. cinerea. Moreover, genetic analysis demonstrated that NPC phase separation plays an important role in plant defense against fungal and bacterial infection as well as insect attack. These findings reveal that phase separation of the NPC central barrier serves as an important mechanism to mediate nucleocytoplasmic transport of immune regulators and activate plant defense against a broad range of biotic stresses.

In situ preparation of PNIPAM biphasic hydrogels

N-isopropylacrylamide (NIPAM) monomer undergoes phase separation in water at very high concentrations, which offers striking opportunities to engineer biphasic hydrogel systems. However, this is a metastable state and NIPAM evolves to crystallize. Besides, the concentration of NIPAM in the lower phase is very small. In this work we show that the aforementioned limitations can be overcome by the incorporation of selected components. Thus, the addition of acrylic acid enables accessing remarkably stable biphasic NIPAM-water systems, as corroborated by DSC (absence of NIPAM crystallization). The addition of a third monomer, diallyldimethylammonium chloride (DDAC), further allows modifying NIPAM partition notably. The phase-distribution of the components for different monomer mixtures was gauged by 1H NMR. Subsequent polymerization leads to the formation of biphasic hydrogels systems in a single step, whose phase morphology and temperature response (swelling response) has been characterized by SEM. This methodology also permits adjusting the crosslinker partition as a function of its nature, which can be used to tune the swelling behaviour of each phase for a certain composition.

Effect of LNR-g-MMA on the Mechanical Properties and Lifetime Estimation of PLA/PP Blends.

Polylactide (PLA) polymer, polypropylene (PP) polymer, and a PLA/PP (70:30 wt%) blend, with liquid natural rubber-graft-methy methacrylate (LNR-g-MMA) of 0.0, 2.5, 5.0, and 10.0 phr as compatibilizers, were prepared by internal mixing and compression molding. The effect of LNR-g-MMA content on the morphology, mechanical properties, water absorption, thermal degradation, and a lifetime of blends based on PLA and PP was investigated. Scanning electron microscopy (SEM) revealed that the PLA/PP blend underwent phase separation, and the presence of LNR-g-MMA in the PLA/PP blend showed a more homogenized and refined blend morphology. Hence, the addition of LNR-g-MMA was used as a compatibilizer to induce miscibility in the PLA/PP blend. The values of tensile strength, elongation at break, and impact strength of the polymer blends increased, whereas water absorption values decreased with increased LNR-g-MMA content. Thermal degradation kinetics was studied over a temperature range of 50-800 °C with multiple heating rates. The results demonstrated that the thermal stability of blends without LNR-g-MMA was greater than that of blends with LNR-g-MMA and that the thermal stability decreased with increasing LNR-g-MMA content. The activation energy (Ea) was calculated by using the Kissinger-Akahira-Sunose method. The Ea value of PLA was much lower than that of PP, and incorporating PP in the PLA matrix increased the Ea. The addition of LNR-g-MMA to the PLA/PP blend decreased the Ea. The lifetime of PLA/PP blends was reduced with the addition of LNR-g-MMA.

Phase separation of α-crystallin-GFP protein and its implication in cataract disease

Cataract, the leading cause of blindness worldwide, is caused by crystallin protein aggregation within the protected lens environment. Phase separation has been implicated as an important mechanism of protein aggregation diseases, such as neurodegeneration. Similarly, cataract has been proposed to be a protein condensation disease in the last century. However, whether crystallin proteins aggregate via a phase separation mechanism and which crystallin protein initiates the aggregation remain unclear. Here, we showed that all types of crystallin-GFP proteins remain soluble under physiological conditions, including protein concentrations, ion strength, and crowding environments. However, in age or disease-induced aberrant conditions, α-crystallin-GFP, including αA- and αB-crystallin-GFP, but not other crystallin-GFP proteins, undergo phase separation in vivo and in vitro. We found that aging-related changes, including higher crystallin concentrations, increased Na+, and decreased K+ concentrations, induced the aggregation of α-crystallin-GFP. Furthermore, H2O2, glucose, and sorbitol, the well-known risk factors for cataract, significantly enhanced the aggregation of αB-crystallin-GFP. Taken together, our results revealed that α-crystallin-GFP forms aggregates via a phase transition process, which may play roles in cataract disease. Opposite to the previously reported function of enhancing the solubility of other crystallin, α-crystallin may be the major aggregated crystallin in the lens of cataract patients.

A Thermodynamic Model for the Insertion Electrochemistry of Battery Cathodes

AbstractInvited for this issue's Front Cover is the group of Prof. Dr. Uwe Schröder. The cover picture shows a crystal layer of nickel hydroxide that undergoes phase separation during electrochemical ion deinsertion (oxidation) and the corresponding modelled cyclic voltammogram. Read the full text of the Research Article at 10.1002/celc.202201118.

Front Cover: A Thermodynamic Model for the Insertion Electrochemistry of Battery Cathodes (ChemElectroChem 7/2023)

The Front Cover illustrates a crystal layer of nickel hydroxide that undergoes phase separation during electrochemical ion deinsertion (oxidation) and its corresponding cyclic voltammogram (CV). This phase separation phenomenon is revealed by a thermodynamic modelling of solid-solution and phase-separation reaction modes in battery cathodes. Furthermore, according to this model, the hysteresis observed during charge-discharge in the CV curves results from this phase separation which itself is a consequence of large radius changes in the crystal's central metal ion. More information can be found in the Research Article by K. Malaie, F. Scholz and U. Schröder.

ATM-SPARK: A GFP phase separation-based activity reporter of ATM.

The kinase ataxia telangiectasia mutated (ATM) plays a key role in the DNA damage response (DDR). It is thus essential to visualize spatiotemporal dynamics of ATM activity during DDR. Here, we designed a robust ATM activity reporter based on phosphorylation-inducible green fluorescent protein phase separation, dubbed ATM-SPARK (separation of phases-based activity reporter of kinase). Upon ATM activation, it undergoes phase separation via multivalent interactions, forming intensely bright droplets. The reporter visualizes spatiotemporal dynamics of endogenous ATM activity in living cells, and its signal is proportional to the amount of DNA damage. ATM-SPARK also enables high-throughput screening of biological and small-molecule regulators. We identified the protein phosphatase 4 that blocks ATM activity. We also identified BGT226 as a potent ATM inhibitor with a median inhibitory concentration of~3.8 nanomolars. Furthermore, BGT226 sensitizes cancer cells to the radiomimetic drug neocarzinostatin, suggesting that BGT226 might be combined with radiotherapeutic treatment. ATM-SPARK achieves large dynamic range, bright fluorescence, and simple signal pattern.

Disrupting the phase separation of KAT8–IRF1 diminishes PD-L1 expression and promotes antitumor immunity

Immunotherapies targeting the PD-1/PD-L1 axis have become first-line treatments in multiple cancers. However, only a limited subset of individuals achieves durable benefits because of the elusive mechanisms regulating PD-1/PD-L1. Here, we report that in cells exposed to interferon-γ (IFNγ), KAT8 undergoes phase separation with induced IRF1 and forms biomolecular condensates to upregulate PD-L1. Multivalency from both the specific and promiscuous interactions between IRF1 and KAT8 is required for condensate formation. KAT8–IRF1 condensation promotes IRF1 K78 acetylation and binding to the CD247 (PD-L1) promoter and further enriches the transcription apparatus to promote transcription of PD-L1 mRNA. Based on the mechanism of KAT8–IRF1 condensate formation, we identified the 2142–R8 blocking peptide, which disrupts KAT8–IRF1 condensate formation and consequently inhibits PD-L1 expression and enhances antitumor immunity in vitro and in vivo. Our findings reveal a key role of KAT8–IRF1 condensates in PD-L1 regulation and provide a competitive peptide to enhance antitumor immune responses.

Multistep Crystallization of Ge‐Rich GST Unveiled by In Situ synchrotron X‐ray diffraction and (scanning) transmission electron microscopy

Among many phase change materials, Ge‐rich GeSbTe (GST) alloys are of considerable interest due to their high thermal stability, a specification required for the next generation of embedded digital memories. This stability results from the fact that these alloys do not crystallize congruently but experience phase separation forming Ge and GST‐225 nanocrystals upon crystallization. However, the details of the crystallization process remain unclear. Combining in situ X‐ray diffraction studies during isothermal annealing and ex situ (scanning) transmission electron microscopy ((S)TEM) observations, the successive phases through which these alloys crystallize are identified. At low temperature (310 °C), the homogeneous amorphous material undergoes phase separation during wherein small regions of different Ge contents are formed. After a long incubation time, Pnma GeTe embryos first crystallize and trigger the heterogeneous crystallization of the Ge cubic phase. While the Ge phase progressively builds up through the addition of new small Ge crystals, cubic GeTe forms. At this point, the microstructure ceases to evolve, and Sb is still dispersed and contained within some remaining amorphous matrix surrounding Ge and GeTe crystals. Higher annealing temperatures (typically 400 °C) are needed to force Sb to diffuse and get incorporated into the GeTe grains to form cubic Ge2Sb2Te5.

Phase separation of Hippo signalling complexes

The Hippo pathway was originally discovered to control tissue growth in Drosophila and includes the Hippo kinase (Hpo; MST1/2 in mammals), scaffold protein Salvador (Sav; SAV1 in mammals) and the Warts kinase (Wts; LATS1/2 in mammals). The Hpo kinase is activated by binding to Crumbs‐Expanded (Crb‐Ex) and/or Merlin‐Kibra (Mer‐Kib) proteins at the apical domain of epithelial cells. Here we show that activation of Hpo also involves the formation of supramolecular complexes with properties of a biomolecular condensate, including concentration dependence and sensitivity to starvation, macromolecular crowding, or 1,6‐hexanediol treatment. Overexpressing Ex or Kib induces formation of micron‐scale Hpo condensates in the cytoplasm, rather than at the apical membrane. Several Hippo pathway components contain unstructured low‐complexity domains and purified Hpo‐Sav complexes undergo phase separation in vitro. Formation of Hpo condensates is conserved in human cells. We propose that apical Hpo kinase activation occurs in phase separated “signalosomes” induced by clustering of upstream pathway components.

Densely Packed Fiber Electrodes Composed of Liquid Crystalline MXenes for High‐Areal‐Density Supercapacitors

Because electrode structure is a crucial determinant of supercapacitor performance, there are multiple studies of supercapacitor electrodes with various structures. Here, experimental observations of the diameter‐dependent supercapacitor performance of a liquid crystalline (LC)‐assisted MXene fiber‐based electrode produced through a wet‐spinning process is reported. It is shown that Ti3C2Tx nanosheets are highly dispersed in water and do not form aggregates or undergo phase separation. MXene dispersion exhibits the typical shear thinning behavior of liquid crystals above the critical mass fraction. Various MXene fibers derived from needles of different diameters can be produced using one method. LC‐assisted MXene fibers are characterized by a high electrical conductivity of up to 2012.27 S cm−1. Furthermore, the optimized MXene fiber‐based device has an areal capacitance of up to ≈6.82 F cm−2, indicating the utility of LC‐assisted MXene fibers in fiber‐shaped supercapacitor devices. The synthetic method has considerable advantages for applications in high‐performance supercapacitors with both stable device operation and high areal capacitance.

Screen for Modulation of Nucleocapsid Protein Condensation Identifies Small Molecules with Anti-Coronavirus Activity.

Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species. Here, we show that N proteins from all seven human coronaviruses (HCoVs) vary in their tendency to undergo phase separation when expressed in human lung epithelial cells. We developed a cell-based high-content screening platform and identified small molecules that both promote and inhibit condensation of SARS-CoV-2 N. Interestingly, these host-targeted small molecules exhibited condensate-modulatory effects across all HCoV Ns. Some have also been reported to exhibit antiviral activity against SARS-CoV-2, HCoV-OC43, and HCoV-229E viral infections in cell culture. Our work reveals that the assembly dynamics of N condensates can be regulated by small molecules with therapeutic potential. Our approach allows for screening based on viral genome sequences alone and might enable rapid paths to drug discovery with value for confronting future pandemics.

Remodeling of Biomembranes and Vesicles by Adhesion of Condensate Droplets.

Condensate droplets are formed in aqueous solutions of macromolecules that undergo phase separation into two liquid phases. A well-studied example are solutions of the two polymers PEG and dextran which have been used for a long time in biochemical analysis and biotechnology. More recently, phase separation has also been observed in living cells where it leads to membrane-less or droplet-like organelles. In the latter case, the condensate droplets are enriched in certain types of proteins. Generic features of condensate droplets can be studied in simple binary mixtures, using molecular dynamics simulations. In this review, I address the interactions of condensate droplets with biomimetic and biological membranes. When a condensate droplet adheres to such a membrane, the membrane forms a contact line with the droplet and acquires a very high curvature close to this line. The contact angles along the contact line can be observed via light microscopy, lead to a classification of the possible adhesion morphologies, and determine the affinity contrast between the two coexisting liquid phases and the membrane. The remodeling processes generated by condensate droplets include wetting transitions, formation of membrane nanotubes as well as complete engulfment and endocytosis of the droplets by the membranes.

Multiple polarity kinases inhibit phase separation of F-BAR protein Cdc15 and antagonize cytokinetic ring assembly in fission yeast.

The F-BAR protein Cdc15 is essential for cytokinesis in Schizosaccharomyces pombe and plays a key role in attaching the cytokinetic ring (CR) to the plasma membrane (PM). Cdc15's abilities to bind to the membrane and oligomerize via its F-BAR domain are inhibited by phosphorylation of its intrinsically disordered region (IDR). Multiple cell polarity kinases regulate Cdc15 IDR phosphostate, and of these the DYRK kinase Pom1 phosphorylation sites on Cdc15 have been shown in vivo to prevent CR formation at cell tips. Here, we compared the ability of Pom1 to control Cdc15 phosphostate and cortical localization to that of other Cdc15 kinases: Kin1, Pck1, and Shk1. We identified distinct but overlapping cohorts of Cdc15 phosphorylation sites targeted by each kinase, and the number of sites correlated with each kinases' abilities to influence Cdc15 PM localization. Coarse-grained simulations predicted that cumulative IDR phosphorylation moves the IDRs of a dimer apart and toward the F-BAR tips. Further, simulations indicated that the overall negative charge of phosphorylation masks positively charged amino acids necessary for F-BAR oligomerization and membrane interaction. Finally, simulations suggested that dephosphorylated Cdc15 undergoes phase separation driven by IDR interactions. Indeed, dephosphorylated but not phosphorylated Cdc15 undergoes liquid-liquid phase separation to form droplets in vitro that recruit Cdc15 binding partners. In cells, Cdc15 phosphomutants also formed PM-bound condensates that recruit other CR components. Together, we propose that a threshold of Cdc15 phosphorylation by assorted kinases prevents Cdc15 condensation on the PM and antagonizes CR assembly.

Lipid droplets as flexible substrates for protein phase separation.

Biomolecular condensates have been shown to nucleate the formation of amyloid fibrils for many proteins implicated in neurodegenerative disorders, such as alpha synuclein in Parkinson's disease and fused in sarcoma (FUS) in amyotrophic lateral sclerosis. Within these fibrils, a distinct lipid signal is often observable, and indeed, a membrane environment is favorable for the formation of amyloid fibrils for a variety of biophysical reasons, including an increase in local protein concentration and conformational shift upon membrane binding. How might intracellular lipid substrates contribute to nucleation of phase separation? Here we observe for the first time that intrinsically disordered proteins can undergo phase separation on the surface of lipid droplets. We find that the low complexity domain of FUS (FUSLC) separates into protein-rich and protein-depleted phases at physiological salt (150mM) and protein concentrations (<5μM) when recruited to the surfaces of lipid droplets through a histidine-Ni-NTA headgroup interaction. The number of LDs showing FUSLC phase separation is strongly affected by variations in the protein and salt concentration, and it follows the trend seen previously with FUSLC droplets in solution. Further, these phase-separated patches demonstrate molecular mobility when assessed using fluorescence recovery after photobleaching (FRAP), which is indicative of a liquid-like state. This phenomenon extends to other well-known phase separating domains, such as LAF1-RGG. Overall, these results demonstrate the potential of monolayer substrates to nucleate protein phase separation and could have important implications for the assembly of intrinsically disordered lipid droplet-binding proteins.

RNA secondary structure modulates the co-translational association of hERG1a and 1b mRNAs.

Cardiac IKr is an important repolarizing potassium current of the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG1/KCNH2 gene and differ only in their amino-terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of the subunit's expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells, and human myocardium. New evidence suggests that neither 1a nor 1b proteins are required for the co-translational complex assembly. Thus, we hypothesize that hERG1a and 1b mRNAs’ physico-chemical properties, especially their structure, contribute to their co-translational association. In an in vitro reconstitution assay, hERG1a and 1b mRNA transcribed and labeled with Cy3 and Cy5, respectively, undergo phase separation in isolation. When mixed, the two transcripts modulate each other's morphology as represented by size, shape, and area covered, suggesting they interact. Synonymous mutations predicted to alter hERG1a secondary structure, without affecting channel function, reduce hERG1a and 1b interaction in vitro. Confocal live-cell imaging in HeLa cells transfected with labeled hERG1a and 1b mRNA show condensate morphologies consistent with those seen in vitro. Co-transfection of the in-vitro labeled mRNAs results in spatial overlap of the respective signals as well as translation of hERG1b protein apparently within the same macromolecular complexes, indicating they are translational. In addition, the findings indicate that co-transcription of the mRNAs is not required for their co-translational association. These results suggest that 1a and 1b mRNA physico-chemical properties modulate their association and subsequent co-translation in live cells.

Structure and dynamics of RNA in biomolecular condensate.

Although it is known that RNA undergoes phase separation, the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamic properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of RNAs. Simulations for the trinucleotide repeat RNAs, implicated in neurological disorders, reveal the structures and dynamics of RNA in condensates, which are drastically different from the dilute phase. Interestingly, the interaction network and dynamics in the condensate depend on the RNA sequence, and the droplet is most compact in the case of repeat RNAs. These findings shed light on the mechanisms of condensate formation of RNA repeat sequences, which can be helpful for the understanding of neurological disease onset.