Liquid-liquid Phase Separation Research Articles

Białka ulegające separacji faz u roślin i metody ich badań

Numerous scientific reports in recent years have indicated that liquid-liquid phase separation (LLPS) is an important mechanism allowing plants to identify various biotic and abiotic stresses and respond to them. The following paper reviews the methods currently used to study macromolecular condensates and the function of proteins undergoing LLPS in plants.

Biphasic Coacervation Controlled by Kinetics as Studied by De Novo-Designed Peptides.

Coacervation is generally treated as a liquid-liquid phase separation process and is controlled mainly by thermodynamics. However, kinetics could make a dominant contribution, especially in systems containing multiple interactions. In this work, using peptides of (XXLY)6SSSGSS to tune the charge density and the degree of hydrophobicity, as well as to introduce secondary structures, we evaluated the effect of kinetics on biphasic coacervates formed by peptides with single-stranded oligonucleotides and quaternized dextran at varying pH values. Only in the case where the charge density is constant and the electrostatic interaction is the major driving force for Coacervation is the effect of kinetics negligible. When pH-dependent electrostatic interaction and hydrophobic interaction are involved or the peptides form secondary structures, the Coacervation process is then path-dependent, indicating that the kinetics controls the phase separation process. The Coacervation by combining two different peptides suggests that the peptide with a higher charge density plays a leading role in the early stage, while the cooperation of both peptides takes over afterward. Our work demonstrates that it is normal to observe coacervates with different morphologies and functions due to kinetic control, especially in living cells. Peptides with minimized sequences are a practical approach to reveal the mechanism of Coacervation processes controlled by kinetics.

Phase transitions in chromatin: Mesoscopic and mean-field approaches.

By means of a minimal physical model, we investigate the interplay of two phase transitions at play in chromatin organization: (1) liquid-liquid phase separation within the fluid solvating chromatin, resulting in the formation of biocondensates; and (2) the coil-globule crossover of the chromatin fiber, which drives the condensation or extension of the chain. In our model, a species representing a domain of chromatin is embedded in a binary fluid. This fluid phase separates to form a droplet rich in a macromolecule (B). Chromatin particles are trapped in a harmonic potential to reproduce the coil and globular phases of an isolated polymer chain. We investigate the role of the droplet material B on the radius of gyration of this polymer and find that this radius varies nonmonotonically with respect to the volume fraction of B. This behavior is reminiscent of a phenomenon known as co-non-solvency: a polymer chain in a good solvent (S) may collapse when a second good solvent (here B) is added in low quantity and expands at higher B concentration. In addition, the presence of finite-size effects on the coil-globule transition results in a qualitatively different impact of the droplet material on polymers of various sizes. In the context of genetic regulation, our results suggest that the size of chromatin domains and the quantity of condensate proteins are key parameters to control whether chromatin may respond to an increase in the quantity of chromatin-binding proteins by condensing or expanding.

Hydrogen Bonding-Driven Adaptive Coacervates as Protocells.

Coacervation based on liquid-liquid phase separation (LLPS) has been widely used for the preparation of artificial protocells and to mimic the dynamic organization of membrane-free organelles. Most complex synthetic coacervates are formed through electrostatic interactions but cannot withstand high ionic strength conditions (>0.1 M). Alternative components and driving forces are highly desired for the formation of natural organelles to overcome the drawbacks of traditional coacervates. Herein, hydrogen bonding-driven adaptive coacervates are reported via the complexation of poly(ethylene glycol) (PEG) and tannic acid (TA). The LLPS behavior of these adaptive coacervates is dependent on the concentration and mass ratio of PEG and TA, which can be used to tune the size of coacervates ranging from 70 nm to 10 μm as well as the morphology of isotropic particles and hollow capsules. Coacervates are stable at high ionic concentrations up to 1 M and can serve as protocells to mimic cellular behaviors including metabolism (e.g., nutrient uptake), phagocytosis, and membrane fusion. The reported approach provides a platform for the rational design of hydrogen bonding-driven coacervates with controllable size and morphology, offering potential applications in protocell construction and therapeutic delivery.

Phase boundaries promote chemical reactions through localized fluxes.

One of the hypothesized functions of biomolecular condensates is to act as chemical reactors, where chemical reactions can be modulated, i.e., accelerated or slowed down, while substrate molecules enter and products exit from the condensate. Similarly, the components themselves that take part in the architectural integrity of condensates might be modified by active (energy consuming, non-equilibrium) processes, e.g., by ATPase chaperones or by kinases and phosphatases. In this work, we study how the presence of spatial inhomogeneities, such as in the case of liquid-liquid phase separation, affects active chemical reactions and results in the presence of directional flows of matter, which are one of the hallmarks of non-equilibrium processes. We establish the minimal conditions for the existence of such spatial currents, and we furthermore find that these fluxes are maximal at the condensate interface. These results propose that some condensates might be most efficient as chemical factories due to their interfaces rather than their volumes and could suggest a possible biological reason for the observed abundance of small non-fusing condensates inside the cell, thus maximizing their surface and the associated fluxes.

Staudinger Reaction-Responsive Coacervates for Cytosolic Antibody Delivery and TRIM21-Mediated Protein Degradation.

A low-molecular-weight compound whose structure strikes a fine balance between hydrophobicity and hydrophilicity may form coacervates via liquid-liquid phase separation in an aqueous solution. These coacervates may encapsulate and convoy proteins across the plasma membrane into the cell. However, releasing the cargo from the vehicle to the cytosol is challenging. Here, we address this issue by designing phase-separating coacervates, which are disassembled by the bioorthogonal Staudinger reaction. We constructed and selected triphenylphosphine-based compounds that formed phase-separated coacervates in an aqueous solution. Reacting the coacervates with azides resulted in microdroplet dissolution, so they received the name Staudinger Reaction-Responsive Coacervates, SR-Coa. SR-Coa could encapsulate proteins, including antibodies, and translocate them across the plasma membrane into the cell. Further treatment of the cell with ethyl azidoacetate induced the cargo dispersion from the puncta to the cytosolic distribution. We showcased an application of the SR-Coa/ethyl azidoacetate system in facilitating the translocation of the EGFR/antibody complex into the cell, which induced EGFR degradation via the TRIM21-dependent pathway both in vitro and in vivo. Besides the membrane protein EGFR, this system could also degrade endogenous protein EZH2. Taken together, here we report a strategy of controlling molecular coacervates by a bioorthogonal reaction in the cell for cytosolic protein delivery and demonstrate its use in promoting targeted protein degradation via the proteasome-dependent pathway.

Coacervate vesicles assembled by liquid-liquid phase separation improve delivery of biopharmaceuticals.

Vesicles play critical roles in cellular materials storage and signal transportation, even in the formation of organelles and cells. Natural vesicles are composed of a lipid layer that forms a membrane for the enclosure of substances inside. Here we report a coacervate vesicle formed by the liquid-liquid phase separation of cholesterol-modified DNA and histones. Unlike a phospholipid-based membrane-bounded vesicle, a coacervate vesicle lacks a membrane structure on the surface and is organized with a high-density liquid layer and a water-filled cavity. Through a straightforward coacervation process, we demonstrate that various biological agents, including virus particles, mRNA, cytokines and peptides, can be innocuously and directly enriched in the liquid phase. In contrast to the droplet-like coacervates that are prone to aggregation challenges, coacervate vesicles display superior kinetic stability, positioning them as a versatile delivery vehicle for biopharmaceuticals. We validate that incorporating oncolytic viruses into these coacervate vesicles endows them with potent oncolytic efficacy and elicits robust anti-tumour immune responses in mouse models.

Benchmarking residue-resolution protein coarse-grained models for simulations of biomolecular condensates.

Intracellular liquid-liquid phase separation (LLPS) of proteins and nucleic acids is a fundamental mechanism by which cells compartmentalize their components and perform essential biological functions. Molecular simulations play a crucial role in providing microscopic insights into the physicochemical processes driving this phenomenon. In this study, we systematically compare six state-of-the-art sequence-dependent residue-resolution models to evaluate their performance in reproducing the phase behaviour and material properties of condensates formed by seven variants of the low-complexity domain (LCD) of the hnRNPA1 protein (A1-LCD)-a protein implicated in the pathological liquid-to-solid transition of stress granules. Specifically, we assess the HPS, HPS-cation-π, HPS-Urry, CALVADOS2, Mpipi, and Mpipi-Recharged models in their predictions of the condensate saturation concentration, critical solution temperature, and condensate viscosity of the A1-LCD variants. Our analyses demonstrate that, among the tested models, Mpipi, Mpipi-Recharged, and CALVADOS2 provide accurate descriptions of the critical solution temperatures and saturation concentrations for the multiple A1-LCD variants tested. Regarding the prediction of material properties for condensates of A1-LCD and its variants, Mpipi-Recharged stands out as the most reliable model. Overall, this study benchmarks a range of residue-resolution coarse-grained models for the study of the thermodynamic stability and material properties of condensates and establishes a direct link between their performance and the ranking of intermolecular interactions these models consider.

Modeling and Elucidating the Behavior of a Thermoresponsive LCST Ionic Liquid.

Desalination of seawater by forward osmosis is a technology potentially able to address the global water scarcity problem. The major challenge limiting its widespread practical application is the design of a draw solute that can be separated from water by an energetically efficient process and then reused for the next cycle. Recent experiments demonstrate that a promising draw solute for forward-osmosis desalination is tetrabutylphosphonium 2,4,6-trimethylbenzenesulfonate ([P4444][TMBS]). When mixed with water, this ionic liquid (IL) is thermoresponsive and exhibits a lower critical solution temperature (LCST), above which it phase-separates into an IL-rich phase and a water-rich phase. Elucidating the physical mechanism of the liquid-liquid phase separation, as well as rationally designing optimized derivatives, necessitates an accurate model to describe this and related ILs. In this paper, we resort to explicit-solvent all-atom molecular dynamics simulations and adopt AMBER-based force-field parameters for the cation whose partial charges were assigned by the RESP fitting procedure. Utilizing the same methodology, we parametrize the anion. The simulations' results indicate the IL/water mixture, at the experimental critical composition, can unambiguously phase-separate only when the partial charges of the ions are scaled down. Nevertheless, the best-performing charge scaling factor is found to be 0.95, a value much milder than those reported for ILs in neat phases. This can be explained by a diminished charge transfer, or induced dipoles, within the ions when the IL is in a mixture with water. With this charge scaling, the simulations reproduce well the LCST composition-temperature phase diagram, albeit overestimation of the critical temperature by 10 K. In particular, very good agreement is obtained for the composition of the two segregated phases. Estimation of viscosity points to IL/water mixture that is almost twice as viscous in simulations than that reported experimentally. Furthermore, we analyze changes in energy between different components in the mixture and find that the driving force for phase separation is, at least, enthalpic. Structural analyses of the ions and their interactions with water molecules corroborate the importance of the latter in mediating structural organizations of the anions, as well as in strengthening the interactions between the cations.

Establishment of liquid-liquid phase separation-related prognostic model in lung adenocarcinoma and systematic analysis of its clinical significance.

To detect the prognostic importance of liquid-liquid phase separation (LLPS) in lung adenocarcinoma. The gene expression files, copy number variation data, and clinical data were downloaded from The Cancer Genome Atlas cohort. LLPS-related genes were acquired from the DrLLPS website. The prognostic model based on LLPS was constructed by the Cox regression and LASSO regression analyses after the identification of LLPS-related differentially expressed genes (DEGs). Gene Ontology functional and Kyoto Encyclopedia of Genes and Genomes enrichment analysis were performed. The LLPS-related prognostic risk score was validated by GSE31210 and GSE72094. The overall survival of lung adenocarcinoma patients was predicted by plotting a nomogram. The biological features of the high-risk lung adenocarcinoma were evaluated by the CIBERSORT, ESTIMATE, Gene Set Variation Analysis, and Genomics of Drug Sensitivity in Cancer. Reverse transcription-quantitative polymerase chain reaction detected hub gene expression. A total of 91 DEGs were screened out in LLPS, among which 9 genes were discovered as prognostic biomarkers of lung adenocarcinoma. GRIA1, CRTAC1, MAGEA4, and MAPK4 were identified as hub genes by the LASSO Cox regression analysis. High-risk and low-risk groups were divided according to the risk index, with the high-risk group displaying a markedly worse outcome. CRTAC1 expression was significantly decreased, MAGEA4 and MAPK4 expressions were increased, while GRIA1 expression was altered in lung adenocarcinoma cells. Tumor microenvironment, signaling pathway enrichment, and drug sensitivity significantly differed between different risk groups. This work proposed a prognostic tool based on the LLPS-related gene signature to offer prospective and effective biomarkers for lung adenocarcinoma prognosis.

Abiotic catalysis promoted by liquid-liquid phase separation.

Abiotic catalysis promoted by liquid-liquid phase separation.

Catalytic Assembly of Peptides Mediated by Complex Coacervates.

The assembly of peptides is generally mediated by liquid-liquid phase separation, which enables control over assembly kinetics, final structure, and functions of peptide-based supramolecular materials. Modulating phase separation can alter the assembly kinetics of peptides by changing solvents or introducing external fields. Herein, we demonstrate that the assembly of peptides can be effectively catalyzed by complex coacervates. The negatively charged sodium alginate (SA) can form complex coacervates with the positively charged KLVFFAE (Aβ16-22, abbreviated as KE) peptide, thereby lowering the nucleation barrier and promoting the assembly of the peptide. As the binding affinity of SA-KE and the dosage of SA decrease, the system shifts from a relatively inefficient template-induced assembly to a highly efficient catalytic assembly before ultimately reverting to slow spontaneous assembly. Therefore, both the affinity as well as the stoichiometry do not follow the intuitive rule that "more is better", but rather there exists an optimal value that maximizes the rate of assembly.

Probing Interparticle Interaction and Ordering in Silica-Pluronic-Based Solutions and Emulsions by Small-Angle Scattering Techniques.

Introduction of non-DLVO forces by nonionic surfactants brings about fascinating changes in the phase behavior of silica nanosuspensions. We show here that alterations in the interaction and wetting properties of negatively charged silica nanoparticles (Ludox® LS) in the presence of polyethylene oxide-polypropylene oxide-polyethylene oxide-based triblock copolymers called Pluronics lead to the formation of stable o/w Pickering emulsions and interparticle attraction-induced thermoresponsive liquid-liquid phase separations. The results make interesting comparisons with those reported for Ludox® TM nanosuspensions comprising larger silica nanoparticles. Association of these nanosystems with Pluronics occurs through their surface silanol groups. LS nanoparticles with a higher surface-to-volume ratio thus need a higher amount of Pluronics for the onset of interparticle attraction as compared to their TM counterparts. Small-angle X-ray scattering studies reveal that unlike TM nanosuspensions, LS nanosuspensions form Pickering emulsions with the ordering of both Pluronic-coated and bare nanoparticles at the oil-water interface. This could arise due to steric limitations in accommodating large Pluronic molecules between smaller LS nanoparticles, with highly curved surfaces, in closed-packed configurations. Small angle neutron scattering studies show clear signatures of the onset of thermoresponsive intermicellar attraction in these systems as a function of temperature and Pluronic concentration, induced solely by the modulation of non-DLVO steric and hydrophobic interactions, not reported hitherto in charged nanosuspensions. The results give insights into the roles of hydrophilic-lipophilic balance of surfactants and size of silica nanoparticles in determining the phase behaviors of silica-surfactant nanocomposite systems.

Identification of Biomarkers Related to Liquid-Liquid Phase Separation for Ulcerative Colitis Based on Single-Cell and Bulk RNA Transcriptome Sequencing Data.

Liquid-Liquid Phase Separation (LLPS) is a process involved in the formation of established organelles and various condensates that lack membranes; however, the relationship between LLPS and Ulcerative Colitis (UC) remains unclear. This study aimed to comprehensively clarify the correlation between ulcerative colitis (UC) and liquid-liquid phase separation (LLPS). In this study, bioinformatics analyses and public databases were applied to screen and validate key genes associated with LLPS in UC. Furthermore, the roles of these key genes in UC were comprehensively analyzed. Based on the single-cell transcriptomic data of UC obtained from the Gene Expression Omnibus (GEO) database, differences between patients with UC and their controls were compared using the limma package. The single-cell data were then filtered and normalized by the 'Seurat' package and subjected to dimension reduction by the Uniform Manifold Approximation and Projection (UMAP) algorithm. The LLPS-related genes (LLPSRGs) were searched on the Dr- LLPS website to obtain cross-correlated genes, which were scored using the ssGSEA algorithm. Next, functional enrichment, interaction network, immune landscape, and diagnostic and drug prediction of the LLPSRGs were comprehensively explored. Finally, the results were validated using external datasets and quantitative real-time PCR (qRT-PCR). A total of eight cell types in UC were classified, namely, fibroblasts, macrophages, endothelial cells, neutrophils, NK cells, B cells, epithelial cells, and T cells. The intersection between differently expressed genes (DEGs) among the eight cell types identified 44 key genes, which were predominantly enriched in immune- and infection-related pathways. According to receiver operating characteristic (ROC) curves, PLA2G2A, GZMK, CD69, HSP90B1, and S100A11 reached an AUC value of 0.94, 0.95, 0.86, 0.89, and 0.93, respectively. Drug prediction revealed that decitabine, tetrachlorodibenzodioxin, tetradecanoylphorbol acetate, thapsigargin, and cisplatin were the potential small molecular compounds for PLA2G2A, GZMK, CD69, HSP90B1, and S100A11. Immune cell infiltration analysis demonstrated that the infiltration of CD4 memory T cell activation, macrophage M1, T macrophage M0, neutrophils, and mast cell activation was higher in the UC group than in the normal group. The LLPSRGs play crucial roles in UC and can be used as prognostic and diagnostic markers for UC. The current findings contribute to the management of UC.

Artificial metalloenzyme assembly in cellular compartments for enhanced catalysis.

Artificial metalloenzymes (ArMs) integrated within whole cells have emerged as promising catalysts; however, their sensitivity to metal centers remains a systematic challenge, resulting in diminished activity and turnover. Here we address this issue by inducing in cellulo liquid-liquid phase separation through a self-labeling fusion protein, HaloTag-SNAPTag. This strategy creates membraneless, isolated liquid condensates within Escherichia coli as protective compartments for the assembly of ArMs using the same fusion protein. The approach allows for high ArM loading and stabilization by localizing the ArMs within the phase-separated regions. Consequently, the performance of ArM-based whole-cell catalysts is improved, with a demonstrated turnover per cell of up to 7.1 × 109 for the olefin metathesis reaction. Furthermore, we apply this to an engineered E. coli system in live mice, where host bacterial cells confine the metal catalytic species, and in a mouse colorectal cancer model, where ArM-containing whole-cell catalysts mediate concurrent reactions to activate prodrugs.

Halogenated Cholesterol Alters the Phase Behavior of Ternary Lipid Membranes.

Eukaryotic plasma membranes exhibit nanoscale lateral lipid heterogeneity, a feature that is thought to be central to their function. Studying these heterogeneities is challenging since few biophysical methods are capable of detecting domains at submicron length scales. We recently showed that cryogenic electron microscopy (cryo-EM) can directly image nanoscale liquid-liquid phase separation in extruded liposomes due to its ability to resolve the intrinsic thickness and electron density differences of ordered and disordered phases. However, the intensity contrast between these phases is poor compared with conventional fluorescence microscopy and is thus both a limiting factor and a focal point for optimization. Because the fundamental source of intensity contrast is the spatial variation in electron density within the bilayer, lipid modifications aimed at selectively increasing the electron density of one phase might enhance the ability to resolve coexisting phases. To this end, we investigated model membrane mixtures of DPPC/DOPC/cholesterol in which one hydrogen of cholesterol's C19 methyl group was replaced by an electron-rich halogen atom (either bromine or iodine). We characterized the phase behavior as a function of composition and temperature using fluorescence microscopy, Förster resonance energy transfer, and cryo-EM. Our data suggest that halogenated cholesterol variants distribute approximately evenly between liquid-ordered and liquid-disordered phases and are thus ineffective at enhancing the intensity difference between them. Furthermore, replacing more than half of the native cholesterol with halogenated cholesterol variants dramatically reduces the size of the membrane domains. Our results reinforce how small changes in the sterol structure can have a large impact on the lateral organization of membrane lipids.

Phase separation of initiation hubs on cargo is a trigger switch for selective autophagy.

Autophagy is a key cellular quality control mechanism. Nutrient stress triggers bulk autophagy, which nonselectively degrades cytoplasmic material upon formation and liquid-liquid phase separation of the autophagy-related gene 1 (Atg1) complex. In contrast, selective autophagy eliminates protein aggregates, damaged organelles and other cargoes that are targeted by an autophagy receptor. Phase separation of cargo has been observed, but its regulation and impact on selective autophagy are poorly understood. Here, we find that key autophagy biogenesis factors phase separate into initiation hubs at cargo surfaces in yeast, subsequently maturing into sites that drive phagophore nucleation. This phase separation is dependent on multivalent, low-affinity interactions between autophagy receptors and cargo, creating a dynamic cargo surface. Notably, high-affinity interactions between autophagy receptors and cargo complexes block initiation hub formation and autophagy progression. Using these principles, we converted the mammalian reovirus nonstructural protein µNS, which accumulates as particles in the yeast cytoplasm that are not degraded, into a neo-cargo that is degraded by selective autophagy. We show that initiation hubs also form on the surface of different cargoes in human cells and are key to establish the connection to the endoplasmic reticulum, where the phagophore assembly site is formed to initiate phagophore biogenesis. Overall, our findings suggest that regulated phase separation underscores the initiation of both bulk and selective autophagy in evolutionarily diverse organisms.

Tunable Bicontinuous Macroporous Cell Culture Scaffolds via Kinetically Controlled Phase Separation.

3D scaffolds enable biological investigations with a more natural cell conformation. However, the porosity of synthetic hydrogels is often limited to the nanometer scale, which confines the movement of 3D encapsulated cells and restricts dynamic cell processes. Precise control of hydrogel porosity across length scales remains a challenge and the development of porous materials that allow cell infiltration, spreading, and migration in a manner more similar to natural ECM environments is desirable. Here, a straightforward and reliable method is presented for generating kinetically-controlled macroporous biomaterials using liquid-liquid phase separation between poly(ethylene glycol) (PEG) and dextran. Photopolymerization-induced phase separation resulted in macroporous hydrogels with tunable pore size. Varying light intensity and hydrogel composition controlled polymerization kinetics, time to percolation, and complete gelation, which defined the average pore diameter (Ø = 1-200 µm) and final gel stiffness of the formed hydrogels. Critically, for biological applications, macroporous hydrogels are prepared from aqueous polymer solutions at physiological pH and temperature using visible light, allowing for direct cell encapsulation. Human dermal fibroblasts in a range of macroporous gels are encapsulated with different pore sizes. Porosity improved cell spreading with respect to bulk gels and allowed migration in the porous biomaterials.

Bioinspired programmable coacervate droplets and self-assembled fibers through pH regulation of monomers.

Phase separation and phase transitions pervade the biological domain, where proteins and RNA engage in liquid-liquid phase separation (LLPS), forming liquid-like membraneless organelles. The misregulation or dysfunction of these proteins culminates in the formation of solid aggregates via a liquid-to-solid transition, leading to pathogenic conditions. To decipher the underlying mechanisms, synthetic LLPS has been examined through complex coacervate formation from charged polymers. Nonetheless, temporal control over phase transitions from prebiotically relevant small organic synthons remains largely unexplored. Herein, we propose utilizing pH modulation to regulate the charge of small molecular building blocks, thereby controlling the LLPS process. Through a bio-inspired, enzyme-mediated pH-regulated reaction, we introduce temporal control over both LLPS and the transition from coacervates to supramolecular polymers. Additionally, by incorporating antagonistic pH modulators, we achieve transient LLPS and further temporal regulation of supramolecular polymer disassembly. Our investigation into pH-regulated LLPS provides a new avenue for exploring the stimuli-responsive, dynamic, and transient nature of LLPS.

Enhanced liquid-liquid phase separation of stress granules in a reconstructed model and their cytoplasmic targeting using a DNA nanodevice.

Biomolecular condensates (BCs) are crucial membraneless organelles formed through the process of liquid-liquid phase separation (LLPS) involving proteins and nucleic acids. These LLPS processes are tightly linked with essential cellular activities. Stress granules (SGs), functioning as cytoplasmic BCs, play indispensable roles in maintaining cellular homeostasis and are implicated in diseases like cancers and neurodegenerative disorders. However, devices that can regulate SG LLPS are lacking. Herein, a triangular prism-shaped DNA nanostructure containing polythymidine (ΔDNA(polyT)) is presented as a nanodevice to investigate the LLPS process of in vitro reconstructed SGs (rSGs), a mixture of marker protein G3BP1 and total RNAs. Our observations reveal that the concentration threshold required for rSG LLPS decreases upon addition of ΔDNA(polyT), suggesting an enhancement in SG LLPS efficiency. It is speculated that ΔDNA(polyT) can concentrate mRNAs onto its surface via polyT hybridization with poly-adenosine sequences (polyA) in mRNAs. This alteration in the spatial distribution of mRNAs subsequently affects the multivalency interactions between G3BP1 and mRNAs. Furthermore, ΔDNA(polyT) exhibits excellent colocalization with cytoplasmic SGs under stressed conditions. This DNA-based nanodevice presents a new artificial approach for the targeted regulation of BC LLPS and holds promise for future studies focusing on BCs.