Promote Phase Separation Research Articles

Efficient β-carotene recovery from surfactant-based aqueous solutions: Advancing from experimental to process simulation

β-carotene is a carotenoid present in the human diet with numerous functions in the body. This work suggests a new platform for efficiently recovering this bioactive compound from aqueous solutions. For the first time, a ternary deep eutectic solvent (DES) is proposed to promote phase separation in aqueous solutions of non-ionic surfactants (Triton X-102 and Tween 20). The influence of temperature on the miscibility regions was investigated from 298.15 K to 308.15 K as a preliminary step. The obtained phase diagrams at three temperatures were accurately correlated using four empirical equations. The extraction capacity of these novel combinations was analysed by means of the Tie-Lines (TL) and their derived magnitudes: Tie-Line Length (TLL) and Slope (S). The reliability of the TLs was ascertained through three empirical equations. Analysis of the data showed that more than 99 % of the model carotene could be extracted using ChCl:U:Gly from a Triton X-102 aqueous solution at 308.15 K. Process simulation using SuperPro Designer confirmed that an effective and sustainable process for recovering β-carotene from algal biomass can be designed using a simple approach.

Poly-GR Impairs PRMT1-Mediated Arginine Methylation of Disease-Linked RNA-Binding Proteins by Acting as a Substrate Sink.

Dipeptide repeat proteins (DPRs) are aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two neurodegenerative diseases characterized by the cytoplasmic mislocalization and aggregation of RNA-binding proteins (RBPs). In particular, arginine (R)-rich DPRs (poly-GR and poly-PR) have been suggested to promiscuously interact with multiple cellular proteins and thereby exert high cytotoxicity. Components of the protein arginine methylation machinery have been identified as modulators of DPR toxicity and/or potential cellular interactors of R-rich DPRs; however, the molecular details and consequences of such an interaction are currently not well understood. Here, we demonstrate that several members of the family of protein arginine methyltransferases (PRMTs) can directly interact with R-rich DPRs in vitro and in the cytosol. In vitro, R-rich DPRs reduce solubility and promote phase separation of PRMT1, the main enzyme responsible for asymmetric arginine-dimethylation (ADMA) in mammalian cells, in a concentration- and length-dependent manner. Moreover, we demonstrate that poly-GR interferes more efficiently than poly-PR with PRMT1-mediated arginine methylation of RBPs such as hnRNPA3. We additionally show by two alternative approaches that poly-GR itself is a substrate for PRMT1-mediated arginine dimethylation. We propose that poly-GR may act as a direct competitor for arginine methylation of cellular PRMT1 targets, such as disease-linked RBPs.

Biosynthesis of High Toughness Poly(3-Hydroxypropionate)-Based Block Copolymers With Poly(D-2-Hydroxybutyrate) and Poly(D-Lactate) Segments Using Evolved Monomer Sequence-Regulating Polyester Synthase.

This study synthesized poly(3-hydroxypropionate) [P(3HP)]-containing polyhydroxyalkanoate (PHA) block copolymers, P(3HP)-b-P[2-hydroxybutyrate (2HB)] and P(3HP)-b-P(D-lactate) (PDLA), using Escherichia coli. The cells expressing an evolved sequence-regulating PHA synthase, PhaCARNDFH, and propionyl-CoA transferase were cultured with the supplementation of the corresponding monomer precursors in the medium. The block structure of P(3HP)-b-PDLA was confirmed by proton nuclear magnetic resonance analysis and solvent fractionation. The molecular weights of the polymers were in the range of 0.8-2.8 × 105. The solvent-cast polymer films were subjected to isothermal treatment to promote phase separation and crystallization and were subsequently melt-quenched to produce an amorphous phase. The melt-quenched P(3HP)-b-P(2HB) film exhibited a high elongation at break (1153%), resulting in a toughness of 181 MJ/m3. The solvent-cast film of P(3HP)-b-65 mol% PDLA exhibited partial elastic deformation, in which the P(3HP) phase functioned as a soft segment. The melt-quenching of the polymer resulted in embrittlement presumably due to the high lactate fraction. Overall, the P(3HP)-based block copolymers exhibited several mechanical properties depending on the higher-order structure of the polymer and the properties of the P(2-hydroxyalkanoate) segments. This study findings show that P(3HP)-b-P(2HB) and P(3HP)-b-PDLA can function excellently if their microstructures are properly controlled.

Energy landscapes for clusters of hexapeptides.

We present the results for energy landscapes of hexapeptides obtained using interfaces to the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) program. We have used basin-hopping global optimization and discrete path sampling to explore the landscapes of hexapeptide monomers, dimers, and oligomers containing 10, 100, and 200 monomers modeled using a residue-level coarse-grained potential, Mpipi, implemented in LAMMPS. We find that the dimers of peptides containing amino acid residues that are better at promoting phase separation, such as tyrosine and arginine, have melting peaks at higher temperature in their heat capacity compared to phenylalanine and lysine, respectively. This observation correlates with previous work on the same uncapped hexapeptide monomers modeled using atomistic potential. For oligomers, we compare the variation in monomer conformations with radial distance and observe trends for selected angles calculated for each monomer. The LAMMPS interfaces to the GMIN and OPTIM programs for landscape exploration offer new opportunities to investigate larger systems and provide access to the coarse-grained potentials implemented within LAMMPS.

Evolutionary analysis of ZAP and its cofactors identifies intrinsically disordered regions as central elements in host-pathogen interactions

The zinc-finger antiviral protein (ZAP) is an innate immunity sensor of non-self nucleic acids. Its antiviral activity is exerted through the physical interaction with different cofactors, including TRIM25, Riplet and KHNYN. Cellular proteins that interact with infectious agents are expected to be engaged in genetic conflicts that often result in their rapid evolution. To test this possibility and to identify the regions most strongly targeted by natural selection, we applied in silico molecular evolution tools to analyze the evolutionary history of ZAP and cofactors in four mammalian groups. We report evidence of positive selection in all genes and in most mammalian groups. On average, the intrinsically disordered regions (IDRs) embedded in the four proteins evolve significantly faster than folded domains and most positively selected sites fall within IDRs. In ZAP, the PARP domain also shows abundant signals of selection, and independent evolution in different mammalian groups suggests modulation of its ADP-ribose binding ability. Detailed analyses of the biophysical properties of IDRs revealed that chain compaction and conformational entropy are conserved across mammals. The IDRs in ZAP and KHNYN are particularly compact, indicating that they may promote phase separation (PS). In line with this hypothesis, we predicted several PS-promoting regions in ZAP and KHNYN, as well as in TRIM25. Positively selected sites are abundant in these regions, suggesting that PS may be important for the antiviral functions of these proteins and the evolutionary arms race with viruses. Our data shed light into the evolution of ZAP and cofactors and indicate that IDRs represent central elements in host-pathogen interactions.

The glycine-rich domain of GRP7 plays a crucial role in binding long RNAs and facilitating phase separation

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7’s RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation.

Fluorescent protein tags affect the condensation properties of a phase-separating viral protein.

Fluorescent protein (FP) tags are extensively used to visualize and characterize the properties of biomolecular condensates despite a lack of investigation into the effects of these tags on phase separation. Here, we characterized the dynamic properties of µNS, a viral protein hypothesized to undergo phase separation and the main component of mammalian orthoreovirus viral factories. Our interest in the sequence determinants and nucleation process of µNS phase separation led us to compare the size and density of condensates formed by FP::µNS to the untagged protein. We found an FP-dependent increase in droplet size and density, which suggests that FP tags can promote µNS condensation. To further assess the effect of FP tags on µNS droplet formation, we fused FP tags to µNS mutants to show that the tags could variably induce phase separation of otherwise noncondensing proteins. By comparing fluorescent constructs with untagged µNS, we identified mNeonGreen as the least artifactual FP tag that minimally perturbed µNS condensation. These results show that FP tags can promote phase separation and that some tags are more suitable for visualizing and characterizing biomolecular condensates with minimal experimental artifacts.

Competition between Self-Assembly and Phase Separation Governs High-Temperature Condensation of a DNA Liquid.

In many biopolymer solutions, attractive interactions that stabilize finite-sized clusters at low concentrations also promote phase separation at high concentrations. Here we study a model biopolymer system that exhibits the opposite behavior, whereby self-assembly of DNA oligonucleotides into finite-sized, stoichiometric clusters tends to inhibit phase separation. We first use microfluidics-based experiments to map a novel phase transition in which the oligonucleotides condense as the temperature increases at high concentrations of divalent cations. We then show that a theoretical model of competition between self-assembly and phase separation quantitatively predicts changes in experimental phase diagrams arising from DNA sequence perturbations. Our results point to a general mechanism by which self-assembly shapes phase boundaries in complex biopolymer solutions.

An amino acid ionic liquid-based biphasic solvent with low viscosity, small rich-phase volume, and high CO2 loading rate for efficient CO2 capture

In order to achieve efficient CO2 capture, a novel biphasic solvent based on diethylenetriamine serine ionic liquid/polyethylene glycol dimethyl ether/water ([DETA][SER]/NHD/H2O) was developed. This study achieved low viscosity by weakening the hydrogen bonding and van der Waals interactions within the pure ionic liquid (IL) by adding NHD and H2O to [DETA][SER]. Additionally, due to the low polarity and fewer hydrogen bonds formed by NHD, it is separated. The formation of a tight hydrogen bond network in the rich-phase after the reaction enables the enrichment of the product. Based on this, the optimal mass ratio of [DETA][SER]/NHD/H2O is determined to be 20 wt%/40 wt%/40 wt%. The viscosity of this solvent is 7.82 mPa·s, with a total absorption load of 1.26 mol·mol−1 IL, where the rich-phase load accounts for 99 % of the total load and occupies only 37 % of the volume. The mechanism of CO2 capture was investigated using 13C NMR, revealing the formation of zwitterions from the reaction of the primary amines on [DETA]+ and [SER]− with CO2. Subsequently, proton transfer and hydrolysis of the carbamate esters were observed. Notably, NHD was found to promote phase separation without participating in chemical reactions. These findings demonstrate the potential of this biphasic solvent system as a high-capacity solution for CO2 capture.

Cyclophilin A supports translation of intrinsically disordered proteins and affects haematopoietic stem cell ageing

Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid–liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing.

Experimental and computational studies on isolation of cardanol from cashew nut shell liquid using diethanolamine‐based deep eutectic solvent at different molar ratios

AbstractBACKGROUNDAmine‐based deep eutectic mixtures composed of choline chloride and a DEA‐based hydrogen‐bond donor (HBD) can be used in various molar ratios such as 1:2, 1:3, 1:4, 1:5, 1:6 and 1:7 for isolation of cardanol from cashew nut shell liquid (CNSL).RESULTSIn this study, green techniques were created to extract cardanol from CNSL by utilizing a deep eutectic solvent (DES) based on diethanolamine with various molar ratios [hydrogen‐bond acceptor (HBA): HBD] of 1:2, 1:3,1:4,1:5, 1:6 and 1:7. To enhance cardanol's solubility in DES, promote phase separation, and increase cardanol yield, ethyl acetate (EA) and isobutyl methyl ketone (IBMK) could be utilized as co‐solvents. At temperatures ranging from 293 to 343.15 K and 1 atm, the density of various molar ratios of DES was determined. Additionally, the prepared DES pH behaviour as well as the relationship between pH and temperature were investigated. All investigated DES temperature increases caused a decrease in pH value. By using thermal gravimetric analysis (TGA) and differential scanning calorimetry (DSC), similar molar ratios of amine‐based DESs were studied for their thermal stability in terms of decomposition temperature, mass loss and melting temperature. The melting temperature and thermal stability were both increased when the HBD increased. The thermal stability of DES, which mostly depends on intermolecular interactions, is greatly influenced by HBD. Finally, at T = 298.15 K and 1 atm, cardanol was extracted from 50 mL CNSL using different volumes of various molar ratios of DES (100 mL, 75 mL, 50 mL and 25 mL) together with 25 mL IBMK and 25 mL EA. The extracted cardanol was evaluated using Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry.CONCLUSIONIt was observed that the yields of cardanol from each prepared molar ratios ratio of 100 mL DES were 96.57%, 95.38% and 92.87%, respectively. In this study, the sigma potential and sigma profile of chosen HBA and HBD were determined using conductor‐like screening model for real solvents (COSMO‐RS), and the interactions between the molecules during the extraction process were analyzed. The findings demonstrated the viability of using the chosen DESs for the polyphenol extraction process. © 2024 Society of Chemical Industry (SCI).

Cyclic and Linear Tetrablock Copolymers Synthesized at Speed and Scale by Lewis Pair Polymerization of a One-Pot (Meth)acrylic Mixture and Characterized at Multiple Levels.

Cyclic block copolymers (cBCP) are fundamentally intriguing materials, but their synthetic challenges that demand precision in controlling both the monomer sequence and polymer topology limit access to AB and ABC block architectures. Here, we show that cyclic ABAB tetra-BCPs (cABAB) and their linear counterpart (lABAB) can be readily obtained at a speed and scale from one-pot (meth)acrylic monomer mixtures, through coupling the Lewis pair polymerization's unique compounded-sequence control with its precision in topology control. This approach achieves fast (<15 min) and quantitative (>99%) conversion to tetra-BCPs of predesignated linear or cyclic topology at scale (40 g) in a one-pot procedure, precluding the needs for repeated chain extensions, stoichiometric addition steps, dilute conditions, and postsynthetic modifications, and/or postsynthetic ring-closure steps. The resulting lABAB and cABAB have essentially identical molecular weights (Mn = 165-168 kg mol-1) and block degrees/symmetry, allowing for direct behavioral comparisons in solution (hydrodynamic volume, intrinsic viscosity, elution time, and refractive indices), bulk (thermal transitions), and film (thermomechanical and rheometric properties and X-ray scattering patterns) states. To further the morphological characterizations, allylic side-chain functionality is exploited via the thiol-ene click chemistry to install crystalline octadecane side chains and promote phase separation between the A and B blocks, allowing visualization of microdomain formation.

Surface Charge Can Modulate Phase Separation of Multidomain Proteins.

Phase separation has emerged as an important mechanism explaining the formation of certain biomolecular condensates. Biological phase separation is often driven by the multivalent interactions of modular protein domains. Beyond valency, the physical features of folded domains that promote phase separation are poorly understood. We used a model system─the small ubiquitin modifier (SUMO) and its peptide ligand, the SUMO interaction motif (SIM)─to examine how domain surface charge influences multivalency-driven phase separation. Phase separation of polySUMO and polySIM was altered by pH via a change in the protonation state of SUMO surface histidines. These effects were recapitulated by histidine mutations, which modulated SUMO solubility and polySUMO-polySIM phase separation in parallel and were quantitatively explained by atomistic modeling of weak interactions among proteins in the system. Thus, surface charge can tune the phase separation of multivalent proteins, suggesting a means of controlling phase separation biologically, evolutionarily, and therapeutically.

Fluorescent protein tagging promotes phase separation and alters the aggregation pathway of huntingtin exon-1

Fluorescent protein tags are convenient tools for tracking the aggregation states of amyloidogenic or phase separating proteins, but the effect of the tags is often not well understood. Here, we investigated the impact of a C-terminal red fluorescent protein (RFP) tag on the phase separation of huntingtin exon-1 (Httex1), an N-terminal portion of the huntingtin protein that aggregates in Huntington's disease. We found that the RFP-tagged Httex1 rapidly formed micron-sized, phase separated states in the presence of a crowding agent. The formed structures had a rounded appearance and were highly dynamic according to electron paramagnetic resonance and fluorescence recovery after photobleaching, suggesting that the phase separated state was largely liquid in nature. Remarkably, the untagged protein did not undergo any detectable liquid condensate formation under the same conditions. In addition to strongly promoting liquid-liquid phase separation, the RFP tag also facilitated fibril formation, as the tag-dependent liquid condensates rapidly underwent a liquid-to-solid transition. The rate of fibril formation under these conditions was significantly faster than that of the untagged protein. When expressed in cells, the RFP-tagged Httex1 formed larger aggregates with different antibody staining patterns compared to untagged Httex1. Collectively, these data reveal that the addition of a fluorescent protein tag significantly impacts liquid and solid phase separations of Httex1 invitro and leads to altered aggregation in cells. Considering that the tagged Httex1 is commonly used to study the mechanisms of Httex1 misfolding and toxicity, our findings highlight the importance to validate the conclusions with untagged protein.

Influence of the temperature and type of macromolecule on phase diagrams of aqueous two-phase systems

Phase diagrams for aqueous two-phase systems formed by L64 + Na2SO3 + H2O, F68 + Na2SO3 + H2O, polyethylene glycol 10,000 g·mol–1 (PEG10k) + Na2SO3 + H2O and PEG 35,000 g·mol–1 (PEG35k) + Na2SO3 + H2O were determined experimentally at temperatures of 278.15, 288.15 and 298.15 K, at 0.09 MPa. The binodal curve is not significantly shifted with varying temperature, indicating a small change on the system enthalpy during the phase separation process, but an increase in the temperature promoted an increase in the slope of tie-line. The effectiveness of macromolecules in promoting phase separation occurs in the following order: PEG35k > PEG10k ≈ F68 > L64. An increase in molar mass requires lower concentrations to induce phase separation, due to the decrease of configurational entropy of the solution. When comparing macromolecules with similar molar masses, PEG10k required slightly lower concentrations than F68 to induce phase segregation. Although PEG10k has a higher molar mass, the structure of F68 contains hydrophobic groups (poly(propylene glycol)) that compensate for its lower molar mass, which generates similar phase segregation induction capabilities. Therefore, new aqueous two-phase systems have been characterized by extending the range of systems available for applications.

The variable domain from dynamin-related protein 1 promotes liquid-liquid phase separation that enhances its interaction with cardiolipin-containing membranes.

Dynamins are an essential superfamily of mechanoenzymes that remodel membranes and often contain a "variable domain" important for regulation. For the mitochondrial fission dynamin, dynamin-related protein 1, a regulatory role for the variable domain (VD) is demonstrated by gain- and loss-of-function mutations, yet the basis for this is unclear. Here, the isolated VD is shown to be intrinsically disordered and undergo a cooperative transition in the stabilizing osmolyte trimethylamine N-oxide. However, the osmolyte-induced state is not folded and surprisingly appears as a condensed state. Other co-solutes including known molecular crowder Ficoll PM 70, also induce a condensed state. Fluorescence recovery after photobleaching experiments reveal this state to be liquid-like indicating the VD undergoes a liquid-liquid phase separation under crowding conditions. These crowding conditions also enhance binding to cardiolipin, a mitochondrial lipid, which appears to promote phase separation. Since dynamin-related protein 1 is found assembled into discrete punctate structures on the mitochondrial surface, the inference from the present work is that these structures might arise from a condensed state involving the VD that may enable rapid tuning of mechanoenzyme assembly necessary for fission.

Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units

Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

RNA-binding protein 14 promotes phase separation to sustain prostate specific antigen expression under androgen deprivation in human prostate cancer

Castration-resistant prostate cancer (CRPC) is a big challenge in managing prostate cancer patients. The androgen receptor (AR) pathway is a major driver even in CRPC under androgen deprivation. The mechanism in maintaining of the AR pathway under androgen deprivation remains elusive. The recent discovery of biomolecular condensate, a membrane-less intracellular construct formed by liquid-liquid phase separation (LLPS), that facilitate molecular assembly, encouraged the re-screening of our previous microarray data list. We selected Rbm14 as a target molecule for further analysis because it works as a coactivator of nuclear receptors as well as it facilitates formation of biomolecular condensates via its intrinsically disordered region. GFP-tagged Rbm14 transfected into HEK293T cells formed droplet-like puncta, which diminished following treatment with 1,6-hexanediol. Droplet-like structures were also observed in immunofluorescence for endogenous RBM14 of PC-3 and DU145 cells. Luciferase assay revealed that Rbm14 enhanced androgen-responsive element (ARE)-mediated reporter activity in all conditions with or without testosterone and AR. Co-immunoprecipitation confirmed the Rbm14–AR interaction. Long non-coding RNAs, including NEAT1, SRA1, and HOTAIR, were also interacted with Rbm14. Small interfering RNAs of NEAT1 reduced ARE-mediated reporter activity, while transfection of SRA1 and HOTAIR enhance the reporter activity. Treatment with 1,6-hexanediol as well as transfection with a dominant-negative splice variant of Rbm14 reduced expression of prostate specific antigen (PSA), a prototype of androgen-regulated gene, in LNCaP, PC-3, and DU145 cells under androgen deprivation. Immunohistochemically, RBM14 expression was substantially upregulated in prostate cancer tissues after androgen deprivation therapy than in untreated tumors. In conclusion, RBM14 is a novel factor involved in maintenance of PSA expression via phase separation under androgen deprivation in prostate cancer.

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N.

Микроэкстракционное концентрирование неионными ПАВ и цветометрическое определение фенола

The extraction of the tungsten blue complex formed by the reaction of phenol with Folin-Ciocalteu reagent (FCR) in the presence of a nonionic surfactant Triton X-100 in alkaline medium using the methodology of micellar extraction based on «clouding point» has been studied for the first time. Aqueous solutions of sodium carbonate and sodium sulfate were used to create pH and as a desalting agent. The latter, having a desalting effect, promotes phase separation in the system under study into a micellar phase saturated with surfactant (Triton X-100) and an aqueous phase depleted in surfactant without additional heating at room temperature. Optimal conditions for micellar phenol extraction were determined: FCR (0,2 n.) – Na2CO3 (6%) – Triton X-100 (2%) – Na2SO4 (3,2%). It is shown that micellar-saturated phases of Triton X-100 effectively extract the analytical form (tungsten blue complex), can be proposed for extraction-spectrophotometric and colorimetric determination of phenol. So, for the spectrophotometric determination of phenol (λmax= 760 nm), the Beer-Bouguer-Lambert law obeys an equation of the form: y = 0,0007x – 0,0079, R2 = 0,997. The range of defined contents is in the interval 7·10-7 – 6·10-5 M. A technique for the colorimetric determination of phenol in aqueous media (channel R) has been developed. The intensity of the chromaticity channel R (IR) linearly depends on pc(phenol) in accordance with the equation: y = 95,0x – 455; R2 = 0,997. The range of defined contents is in the interval 7·10-7 – 1·10-5 M. The profiles of the petal diagrams in the RGB CMYK color coordinates are constructed, the dependences of their area are obtained (S) and perimeter (P) from -lgc : (perimeter (P) y = 150x – 284; R2 = 0,994; area (S) y = 17710x – 66930; R2 = 0,994).