Biology subjects

Banerjee, P. R.

Publications and source records attributed to Banerjee, P. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Competing Molecular Interactions Govern the Dynamical Arrest of G3BP1 Condensates

G3BP1 is a central scaffold of stress granules (SGs). Upon cellular stress, G3BP1 forms complex coacervates with translationally repressed mRNAs and recruits multiple RNA-binding proteins to form reversible biomolecular condensates. Persistent SGs are linked to age-dependent dynamical arrest and impaired disassembly. Here, we employ active and passive nanoscale rheology with optical tweezers to show that G3BP1 condensates evolve from being dominantly viscous fluids to dynamically arrested network glasses characterized by nanoscale caging and elastic memory. Integrating atomistic and coarse-grained simulations with experiments, we find that electrostatic interactions between the oppositely charged intrinsically disordered regions drive condensate ageing. RNA modulates these interactions in a length-and structure-dependent manner, delaying dynamic arrest, whereas Caprin-1 binding to the NTF2L domain has little effect. Together, these findings reveal how competing inter-IDR and IDR-RNA interactions govern condensate ageing and material-state transitions. The findings have broader implications for the regulation of SG dynamics in cells.

biophysics↗

bioRxiv2026

Synthetic Biomolecular Condensates as Tunable Microtubule Assembly Hubs

Phase separation of proteins and nucleic acids (NAs) into nano-to-microscale condensates can regulate biochemical processes, including assembly and organization of cytoskeletal networks such as actin and microtubules. This study examines the functional role of condensate material properties in microtubule assembly. Learning from the sequence grammar of naturally occurring intrinsically disordered regions in microtubule-associated proteins, two-component peptide-NA condensates with programmable material properties were designed. These synthetic condensates catalyze tubulin polymerization into microtubule filaments with tunable outcomes. Tubulin preferentially partitions to the condensate interface and nucleates microtubule assembly. Enhanced tubulin self-assembly produces long filaments that exhibit branching and bundling. Using a minimal stochastic chemo-mechanical model, we show that sequence-encoded condensate viscoelasticity is a tunable element that controls filament morphologies and identifies interfacial rheology as the key regulator of filament growth. Fluorescence recovery after photobleaching experiments support this model, revealing a direct correlation between interfacial tubulin mobility and condensate-directed microtubule assembly. Distinct regimes emerge due to competition between bulk adsorption and lateral diffusion of tubulin at the condensate interface, which determines whether filament tips grow or stall. Since dynamic microtubule assembly and restructuring are essential for various cellular functions, this work highlights a critical role of condensate interfacial rheology in cytoskeletal organization.

biophysics↗

bioRxiv2026

Multiscale Physical Effects of CpG Methylation on DNA Mechanics, Nucleosome Wrapping, and Chromatin Condensates

DNA methylation at CpG dinucleotides is a central epigenetic modification linked to transcriptional repression and heterochromatin formation, yet its physical impact on chromatin organization remains incompletely understood. Here, we integrate single-molecule force spectroscopy, FRAP, and nanorheology to quantify how CpG methylation alters chromatin mechanics across scales. Optical tweezer-based measurements show that CpG methylation modestly increases DNA contour and persistence lengths, indicating a slightly extended and stiffer DNA helix. When assembled into nucleosomes, CpG methylation weakens both outer-and inner turn wrapping, reducing unwrapping forces and per-nucleosome unwrapping energy. On a higher-order assembly scale, although CpG methylated and unmethylated chromatin fibers display similar thresholds for salt-induced phase separation, methylated condensates exhibit markedly reduced internal mobility: FRAP reveals slower chromatin exchange, nanorheology shows increased viscoelasticity and longer relaxation times. Finally, CpG methylation enhances HP1-chromatin interactions, lowering the HP1 concentration required for condensation, despite no change in HP1 binding to naked DNA. Together, these results show that CpG methylation remodels chromatin mechanics by tuning DNA rigidity, nucleosome wrapping energetics, and condensate material properties, providing a multiscale physical framework for how DNA methylation contributes to heterochromatin.

biochemistry↗

bioRxiv2025

Viscoelasticity and interface properties of multi-component condensates govern protein sequestration and suppression of amyloid formation

Stress granules (SGs) are multi-component biomolecular condensates widely implicated as sites of protein aggregation by virtue of the high concentrations of amyloidogenic RNA-binding proteins they contain. This model, in which SGs are viewed as crucibles for amyloid formation, has not been rigorously tested. Here, we employed twelve multi-component protein-nucleic acid condensate systems as SG-mimics with diverse physicochemical features. Utilizing three fibril-forming proteins, hnRNPA1, Tau, and FUS, which are concentrated more than 50-fold in condensates, we report that multi-component biomolecular condensates robustly suppress, rather than promote, amyloid formation. Multiscale experimental analyses, including quantitative kinetic measurements, rheology, and microscopy, combined with computational modelling, reveal that condensates serve as sinks for soluble protein, and fibrils form in the dilute phase, although interfaces can promote nucleation. Three key physicochemical properties of condensates govern suppression of fibril formation: First, condensate-mediated sequestration lowers the concentration of fibril-forming proteins in the dilute phase. Second, condensate viscoelasticity constrains efflux-driven fibril growth in the dilute phase. And third, dilution of fibril-forming proteins at condensate interfaces mitigates fibril nucleation. The sink potential of SG-mimics is recapitulated in G3BP1-RNA condensates and SGs reconstituted in mammalian cell lysate, suggesting that SGs may have evolved to suppress stress-induced protein aggregation.

biophysics↗

bioRxiv2025

Intrinsically Disordered Protein Coating for Oral Delivery of Peptide Drugs

AbstractAdvancing oral delivery of peptide therapeutics requires innovative materials that overcome gastrointestinal barriers. We introduce the first engineered synthetic intrinsically disordered protein (SynIDP) that self-assembles into an enteric coating, encapsulating peptide drugs to enhance gastric acid resistance and intestinal targeting. This SynIDP recapitulates the molecular design principles and phase transitions of native IDPs to exhibit temperature-controlled condensation and pH-controlled solidification--both transitions being reversible and precisely tuned to intestinal cues. Through detailed analysis of the kinetics of the liquid-to-solid phase transition, we achieve control over the nano-to-microscale morphology of the protein coating, optimizing drug encapsulation and protection. The coating protects peptide-based weight loss drugs for over 60 minutes in simulated gastric conditions, then dissolves to release the active compound. Oral delivery to obese mice results in more consistent weight loss compared to the unencapsulated drug. This modular protein-based coating is a promising platform technology for enhancing oral peptide drug delivery and improving patient compliance.

bioengineering↗

bioRxiv2025

Histone H3 tail charge patterns govern nucleosome condensate formation and dynamics

Emerging models of nuclear organization suggest that chromatin forms functionally distinct microenvironments through phase separation. As chromatin architecture is organized at the level of the nucleosome and regulated by histone post-translational modifications, we investigated how these known regulatory mechanisms influence nucleosome phase behavior. By systematically altering charge distribution within the H3 tail, we found that the terminal and central regions modulate the phase boundary and tune nucleosome condensate viscosity differentially, as revealed by microscopy-based assays, microrheology, and simulations. Nuclear magnetic resonance relaxation experiments revealed that H3 tails remain dynamically mobile within condensates, and their mobility correlates with condensate viscosity. These results demonstrate that the number, identity, and spatial arrangement of basic residues in the H3 tail critically regulate nucleosome phase separation. Our findings support a model in which nucleosomes, through their intrinsic properties and modifications, actively shape the local chromatin microenvironment--providing new insight into the histone language in chromatin condensates.

biophysics↗

bioRxiv2025

Decoupling Phase Separation and Fibrillization Preserves Activity of Biomolecular Condensates

Age-dependent transition of metastable, liquid-like protein condensates to amyloid fibrils is an emergent phenomenon of numerous neurodegeneration-linked protein systems. A key question is whether the thermodynamic driving forces underlying reversible phase separation and maturation to irreversible amyloids are distinct and separable. Here, we address this question using an engineered version of the microtubule-associated protein Tau, which forms biochemically active condensates. Liquid-like Tau condensates exhibit rapid aging to amyloid fibrils under quiescent, cofactor-free conditions. In particular, the Tau condensate interface promotes fibril nucleation, thereby impairing condensate activity in recruiting tubulin and catalyzing microtubule assembly. Remarkably, a small molecule metabolite, L-arginine, selectively impedes condensate-to-fibril transition without perturbing phase separation in a valence and chemistry-specific manner. By enhancing condensate viscoelasticity, L-arginine counteracts age-dependent decline in the biochemical activity of Tau condensates. These results provide a proof-of-principle demonstration that small molecule metabolites can reinforce the metastability of protein condensates against a liquid-to-amyloid transition, thereby preserving condensate function.

biophysics↗

bioRxiv2025

The Critical Role of the 2'-OH group in Phase Separation and Percolation Transitions of RNA

Mg2+ ion-dependent RNA liquid-liquid phase separation with lower critical solution temperatures is driven by the phosphate backbone and modulated by the solvation property of nucleobases. Here, we report a key role of the 2-OH group of the ribose sugar in RNA condensation in the presence of divalent cations. We show that 2-deoxyribose inhibits nucleic acid phase separation and suppresses the intra-condensate networking transition, known as percolation, that underlies condensate dynamical arrest. All-atom simulations reveal increased solvation and compaction of single-stranded DNA compared to RNA, suggesting an unintuitive role of chain flexibility in modulating heat-induced nucleic acid phase separation and percolation transitions. Further, 2-O-methylation (2-O-Me) of RNA, a common sugar modification, lowers the driving force of RNA phase transitions. These results highlight the diverse physicochemical parameters governing nucleic acid phase behavior and suggest how sugar modifications may have evolved to robustly tune the formation and dynamical arrest of RNA condensates.

biophysics↗

bioRxiv2024

Biomolecular Condensates Can Enhance Pathological RNA Clustering

Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant, and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures, with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which accompanies a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.

biophysics↗

bioRxiv2024

Direct computations of viscoelastic moduli of biomolecular condensates

In vitro facsimiles of biomolecular condensates are formed by different types of intrinsically disordered proteins including prion-like low complexity domains (PLCDs). PLCD condensates are viscoelastic materials defined by time-dependent, sequence-specific complex shear moduli. Here, we show that viscoelastic moduli can be computed directly using a generalization of the Rouse model and information regarding intra- and inter-chain contacts that is extracted from equilibrium configurations of lattice-based Metropolis Monte Carlo (MMC) simulations. The key ingredient of the generalized Rouse model is the Zimm matrix that we compute from equilibrium MMC simulations. We compute two flavors of Zimm matrices, one referred to as the single-chain model that accounts only for intra-chain contacts, and the other referred to as a collective model, that accounts for inter-chain interactions. The single-chain model systematically overestimates the storage and loss moduli, whereas the collective model reproduces the measured moduli with greater fidelity. However, in the long time, low-frequency domain, a mixture of the two models proves to be most accurate. In line with the theory of Rouse, we find that a continuous distribution of relaxation times exists in condensates. The single crossover frequency between dominantly elastic versus dominantly viscous behaviors is influenced by the totality of the relaxation modes. Hence, our analysis suggests that viscoelastic fluid-like condensates are best described as generalized Maxwell fluids. Finally, we show that the complex shear moduli can be used to solve an inverse problem to obtain distributions of relaxation times that underlie the dynamics within condensates.

biophysics↗

bioRxiv2023

Diffusiophoresis promotes phase separation and transport of biomolecular condensates

The internal microenvironment of a living cell is heterogeneous and comprises a multitude of organelles with distinct biochemistry. Amongst them are biomolecular condensates, which are membrane-less, phase-separated compartments enriched in system-specific proteins and nucleic acids. The heterogeneity of the cell engenders the presence of multiple spatiotemporal gradients in chemistry, charge, concentration, temperature, and pressure. Such thermodynamic gradients can lead to non-equilibrium driving forces for the formation and transport of biomolecular condensates. Here, we report how ion gradients impact the transport processes of biomolecular condensates on the mesoscale and biomolecules on the microscale. Utilizing a microfluidic platform, we demonstrate that the presence of ion concentration gradients can accelerate the transport of biomolecules, including nucleic acids and proteins, via diffusiophoresis. This hydrodynamic transport process allows localized enrichment of biomolecules, thereby promoting the location-specific formation of biomolecular condensates via phase separation. The ion gradients further impart active motility of condensates, allowing them to exhibit enhanced diffusion along the gradient. Coupled with a reentrant phase behavior, the gradient-induced active motility leads to a dynamical redistribution of condensates that ultimately extends their lifetime. Together, our results demonstrate diffusiophoresis as a non-equilibrium thermodynamic force that governs the formation and transport of biomolecular condensates.

biophysics↗

bioRxiv2023

Heterotypic interactions in the dilute phase can drive co-condensation of prion-like low-complexity domains of FET proteins and mammalian SWI/SNF complex

Prion-like domains (PLDs) are low-complexity protein sequences enriched within nucleic acid-binding proteins including those involved in transcription and RNA processing. PLDs of FUS and EWSR1 play key roles in recruiting chromatin remodeler mammalian SWI/SNF complex to oncogenic FET fusion protein condensates. Here, we show that disordered low-complexity domains of multiple SWI/SNF subunits are prion-like with a strong propensity to undergo intracellular phase separation. These PLDs engage in sequence-specific heterotypic interactions with the PLD of FUS in the dilute phase at sub-saturation conditions, leading to the formation of PLD co-condensates. In the dense phase, homotypic and heterotypic PLD interactions are highly cooperative, resulting in the co-mixing of individual PLD phases and forming spatially homogeneous co-condensates. Heterotypic PLD-mediated positive cooperativity in protein-protein interaction networks is likely to play key roles in the co-phase separation of mSWI/SNF complex with transcription factors containing homologous low-complexity domains.

biophysics↗

bioRxiv2023

Sequence-encoded grammars determine material properties and physical aging of protein condensates

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

biophysics↗

bioRxiv2022

Determinants of Viscoelasticity and Flow Activation Energy in Biomolecular Condensates

The form and function of biomolecular condensates, which are phase-separated intracellular granules of proteins and RNAs, are regulated by their material and dynamical properties. Emerging reports suggest that biomolecular condensates are viscoelastic network fluids, and the primary sequence and structure of the constituent biopolymers govern their bulk fluid phase properties. Here, we employ a multi-parametric approach to dissect the molecular determinants of condensate viscoelasticity by studying a series of condensates formed by engineered multivalent arginine-rich polypeptides and single-stranded DNA. By measuring the terminal relaxation time of the condensate network through optical tweezer-based microrheology and the activation energy of viscous flow through temperature-controlled video particle tracking, we show that condensate viscoelasticity is controlled by two distinct factors - sequence-encoded inter-chain interactions of associative polymers and entropic factors emerging from their intrinsic polymer properties such as the chain length. The biomolecular diffusion in the dense phase shows a strong dependence on the flow activation energy, indicating that the intra-condensate transport properties are primarily reaction-dominant. These results provide a glimpse of the multifaceted control of viscoelasticity and transport properties within biomolecular condensates. Flow activation energy measurement of single and multicomponent condensates by thermo-rheology provides a direct route to quantify inter-chain interactions in the dense phase and dissect the roles of chain entropy and valence in dictating the viscoelastic behavior of biomolecular condensates.

biophysics↗

bioRxiv2022

RNAs undergo phase transitions with lower critical solution temperatures

Co-phase separation of RNAs and RNA-binding proteins is thought to drive the biogenesis of ribonucleoprotein granules. RNAs can also undergo phase transitions in the absence of proteins. However, the physicochemical driving forces of protein-free, RNA-driven phase transitions remain unclear. Here, we report that RNAs of various types undergo phase transitions with system-specific lower critical solution temperatures (LCSTs). This entropically-driven phase behavior requires Mg2+ ions and is an intrinsic feature of the phosphate backbone that is modulated by RNA bases. RNA-only condensates can additionally undergo enthalpically favorable percolation transitions within dense phases. This is enabled by a combination of Mg2+-dependent bridging interactions among phosphate groups and RNA base-stacking / base-pairing. Phase separation coupled to percolation can cause dynamical arrest of RNAs within condensates and can suppress the catalytic activity of an RNase P ribozyme. Our work highlights the need to incorporate RNA-driven phase transitions into models for RNP granule biogenesis.

biophysics↗

bioRxiv2021

FUS Oncofusion Protein Condensates Recruit mSWI/SNF Chromatin Remodelers via Heterotypic Interactions Between Prion-like Domains

Fusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family of proteins result from fusion of the prion-like domain (PLD) of FET proteins to the DNA-binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS-DDIT3, a FET oncofusion protein, undergoes PLD-mediated phase separation into liquid-like condensates. Nuclear FUS-DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF is driven by heterotypic PLD-PLD interactions between FUS-DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single-molecule correlative force-fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD-driven co-condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD-DBD domain architecture for global transcriptional reprogramming.

biophysics↗

bioRxiv2021

Programmable Viscoelasticity in Protein-RNA Condensates with Disordered Sticker-Spacer Polypeptides

Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.

biophysics↗

bioRxiv2020

Sequence-encoded and Composition-dependent Protein-RNA Interactions Control Multiphasic Condensate Topologies

Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases-homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.

biophysics↗