Coacervates Forming Coexisting Phases on a Mineral Surface.

Minerals played a crucial role in the chemical evolution of small molecules into biopolymers. Yet, it is still not clear how the minerals are related to the formation and the evolution of protocells on early Earth. In this work, using the coacervate formed by quaternized dextran (Q-dextran) and single-stranded oligonucleotides (ss-oligo) as the protocell model, we systematically studied the phase separation of Q-dextran and ss-oligo on the muscovite surface. Serving as rigid and 2D polyelectrolytes, the muscovite surface can be treated by Q-dextran to become negatively charged, neutral, or positively charged. We observed that Q-dextran and ss-oligo form uniform coacervates on naked and neutral muscovite surfaces, while they form biphasic coacervates containing Q-dextran-rich phases and ss-oligo-rich phases on positively or negatively charged muscovite surfaces that were pretreated by Q-dextran. The evolution of the phases is caused by the redistribution of the components as the coacervate touches the surface. Our study indicates that the mineral surface could be a potential driving force for the formation of protocells with hierarchical structures and desirable functions on prebiotic Earth.

Crowded Environment Regulates the Coacervation of Biopolymers via Nonspecific Interactions.

The membrane-less organelles (MLOs) with subcompartments are formed via liquid-liquid phase separation (LLPS) in the crowded cell interior whose background molecules are up to 400 mg/mL. It is still a puzzle how the background molecules regulate the formation, dynamics, and functions of MLOs. Using biphasic coacervate droplets formed by poly(l-lysine) (PLL), quaternized dextran (Q-dextran), and single-stranded oligonucleotides (ss-oligo) as a model of MLO, we online monitored the LLPS process in Bovine Serine Albumin (BSA) solution up to 200.0 mg/mL. Negatively charged BSA is able to form complex or coacervate with positively charged PLL and Q-dextran and thus participates in the LLPS via nonspecific interactions. Results show that BSA effectively regulates the LLPS by controlling the phase distribution, morphologies, and kinetics. With increasing BSA concentration, the spherical biphasic droplets evolve in sequence into phase-inverted flower-like structure, worm-like chains, network structures, and confined coacervates. Each kind of morphology is formed via its own specific growth and fusion pathway. Our work suggests that MLOs could be controlled solely by the crowded environment and provides a further step toward understanding the life process in cell.

Morphology and Dynamics of Coexisting Phases in Coacervate Solely Controlled by Crowded Environment.

The membraneless organelles (MLOs) play a key role in the cell, yet it is unclear what controls the morphology and dynamics of MLOs in crowded cell medium. Using a biphasic coacervate droplet as a model of MLO, we online monitored the liquid-liquid phase separation process in crowded medium provided by poly(ethylene oxide) (PEO) or dextran. In PEO solution, which has an affinity with the inner phase, the spherical droplets evolve into clusters, networks, and completely phase inverted spheres in sequence with increasing PEO concentration, while in dextran solution, which has an affinity with the outer phase, the coacervates maintain the morphology but vary in phase ratio. Flower-like and even Janus structures are formed in the mixed PEO/dextran medium. Our work demonstrates that MLOs could be controlled solely by the crowded cell medium.

Coacervate Formed by an ATP-Binding Aptamer and Its Dynamic Behavior under Nonequilibrium Conditions.

Although numerous protocell models have been developed to explore the possible pathway of the origin of life on the early earth, few truly fulfill the roles of the DNA/RNA sequence and ATP molecules, which are keys to cell replication and evolution. The ATP-binding aptamer offers an opportunity to combine sequence and energy molecules. In this work, we choose the coacervate droplet as the protocell model and investigate the interaction of the DNA aptamer, poly(l-lysine)(PLL), and ATP under varying conditions. PLL and aptamers form solid precipitates, which spontaneously transform to coacervate droplets as ATP is introduced. The selective uptake and sequestration of exogenous molecules is achieved by the ATP-containing coacervates. As an electric field is applied to expel ATP, the portion of the droplet deficient in ATP becomes solid. The solid/liquid phase ratio is tunable by varying the electric field strength and excitation time. Together with the vacuolization process, a solid head with a soft mouth periodically opening and closing is created. Moreover, the composite coacervate droplet gradually grows as it is treated with an electric field and cannot recover to the original liquid phase after the power is turned off and replenished with ATP. Our work highlights that the proper integration of the DNA sequence, ATP, and energy input could be a powerful strategy for creating a protocell with certain living features.

Liquid-Liquid Phase Separation of Peptide/Oligonucleotide Complexes in Crowded Macromolecular Media.

The membraneless organelles (MLOs) and coacervates of oppositely charged polyelectrolytes are both formed by liquid-liquid phase separation. To reveal how the crowded cell interior regulates the MLOs, we chose the coacervates formed by peptide S5 and single-stranded oligonucleotide (ss-oligo) at 1:1 charge ratio and investigated the phase separation processes in polyacrylamide (PAM) and poly(ethylene oxide) (PEO) media at varying concentrations. Results show that the droplet formation unit is the neutral primary complex, instead of individual S5 or ss-oligo. Therefore, the coacervation process can be described by the classic theory of nucleation and growth. The dynamic scaling relationships show that S5/ss-oligo coacervation undergoes in sequence the heterogeneous nucleation, diffusion-limited growth, and Brownian motion coalescence with time. The inert crowders generate multiple effects, including accelerating the growth of droplets, weakening the electrostatic attraction, and slowing down or even trapping the droplets in the crowder network. The overall effect is that both the size and size distribution of the droplets decrease with increasing crowder concentration, and the effect of PEO is stronger than that of PAM. Our study provides a further step toward a deeper understanding of the kinetics of MLOs in crowded living cells.

Protocells with hierarchical structures as regulated by liquid-liquid and liquid-solid phase separations.

The construction of a protocell with dynamic hierarchical structures via spontaneous phase separation sheds light on the mechanisms of life processes. Taking advantage of the transition from the liquid to solid phase, we built composite droplets with PLL/oligo/oligocomp solid particles randomly distributed inside a PLL/oligo liquid coacervate. The circulation and vacuolization under an electric field drive the particles into a fibrous structure and even clusters. A PLL/oligo/oligocomp solid phase can also form on the interface of the PLL/oligo coacervate, turning the droplet into a vesicular structure.

Fission and Internal Fusion of Protocell with Membraneless "Organelles" Formed by Liquid-Liquid Phase Separation.

Construction of protocells with hierarchical structures and living functions is still a great challenge. Growing evidence demonstrates that the membraneless organelles, which facilitate many essential cellular processes, are formed by RNA, protein, and other biopolymers via liquid-liquid phase separation (LLPS). The formation of the protocell in the early days of Earth could follow the same principle. In this work, we develop a novel coacervate-based protocell containing membraneless subcompartments via spontaneous liquid-liquid phase separation by simply mixing single-stranded oligonucleotides (ss-oligo), quaternized dextran (Q-dextran), and poly(l-lysine) (PLL) together. The resulting biphasic droplet, with PLL/ss-oligo phase being the internal subcompartments and Q-dextran/ss-oligo phase as the surrounding medium, is capable of sequestering and partitioning biomolecules into distinct regions. When the droplet is exposed to salt or Dextranase, the surrounding Q-dextran/ss-oligo phase will be gradually dissociated, resulting in the chaotic movement and fusion of internal subcompartments. Besides, the external electric field at a lower strength can drive the biphasic droplet to undergo a deviated circulation concomitant with the fusion of localized subcompartments, while a high-strength electric field can polarize the whole droplet, resulting in the release of daughter droplets in a controllable manner. Our study highlights that liquid-liquid phase separation of biopolymers is a powerful strategy to construct hierarchically structured protocells resembling the morphology and functions of living cells and provides a step toward a better understanding of the transition mechanism from nonliving to living matter under prebiotic conditions.

Macromolecular Crowding and Confinement Effect on the Growth of DNA Nanotubes in Dextran and Hyaluronic Acid Media.

The dense medium modulates the molecular structure and bioreactions in living cells via both noncovalent interactions and macromolecular crowding and confinement effects. However, the interplay between the volume effect and noncovalent interactions remains unclear. In this work, we studied in detail on how electrostatic interactions influence the crowding and confinement effect by comparing the formation and elongation of DNA nanotubes in branched dextran and charged hyaluronic acid (HA) solution of a broad concentration range, with and without 150 mM NaCl. In all the studied cases, three concentration regimes are identified: a crowding regime, a double-effect regime, and a confinement regime. In the crowding and double-effect regimes, the addition of 150 mM NaCl enhances the assembly of DNA tiles by screening the electrostatic repulsion, and a higher dextran solution is required to confine the DNA assembly into nanotubes. However, the screening effect on the HA network is more than that on the DNA assembly, so DNA tubes formed in HA solution at much lower concentrations. In the confinement regime, the electrostatic interaction exhibits a negligible effect on the DNA assembly in both dextran medium and HA medium. Our study demonstrates that the volume effect and noncovalent interactions are system specific and concentration dependent. Their interplay governs the living processes in crowded cells.

Mass Transport in Coacervate-Based Protocell Coated with Fatty Acid under Nonequilibrium Conditions.

Construction of protocell models from prebiotically plausible components to mimic the basic features or functions of living cells is still a challenge. In this work, we prepare a hybrid protocell model by coating sodium oleate on the coacervate droplet constituted by poly(l-lysine) and oligonucleotide and investigate the transport of different molecules under electric field. Results show that sodium oleate forms a layered viscoelastic membrane on the droplet surface, which is selectively permeable to small, polar molecules, such as oligolysine. As the droplet is stimulated at 10 V cm-1, the oleate membrane slips along the direction of electric field while maintaining its integrity. Most of the molecules are still excluded under such conditions. As repetitive cycles of vacuolization occur at 20 V cm-1, all molecules are internalized and sequestrated in the droplet through their specific pathways except enzyme, which anchors in the oleate membrane and is immune to electric field. Cascade enzymatic reactions are then carried out, and the product generated from the membrane exhibits a time-dependent concentration gradient across the droplet. Our work makes a step toward the nonequilibrium functionalization of synthetic protocells capable of biomimetic operations.

One-step radiation synthesis of agarose/polyacrylamide double-network hydrogel with extremely excellent mechanical properties.

A facile one-step radiation method is first developed to synthesize agarose/polyacrylamide (AG/PAM) double-network (DN) hydrogel. Compared to other synthetic methods of DN hydrogels, our synthesis method endows the resultant AG/PAM DN hydrogel with not only top-level tensile properties with a tensile strength of 1263 ± 59 kPa and an elongation at break of 3406 ± 143%, but also highest compression properties with a compression strength of 140 ± 3 MPa and a fracture compression strain of above 99.9%. An expanding-necking phenomenon during compression process of AG/PAM DN hydrogel were observed. We propose a chain pushing-in model to interpret the energy dissipation mechanism accounting for the super-compressibility of AG/PAM DN hydrogel. This novel radiation synthesis strategy provides an insight into the development of DN hydrogels with extremely excellent mechanical properties.

One-Pot Synthesis of a Double-Network Hydrogel Electrolyte with Extraordinarily Excellent Mechanical Properties for a Highly Compressible and Bendable Flexible Supercapacitor.

High-performance hydrogel electrolytes play a crucial role in flexible supercapacitors (SCs). However, the unsatisfactory mechanical properties of widely used polyvinyl alcohol-based electrolytes greatly limit their use in the flexible SCs. Here, a novel Li2SO4-containing agarose/polyacrylamide double-network (Li-AG/PAM DN) hydrogel electrolyte was synthesized by a heating-cooling and subsequent radiation-induced polymerization and cross-linking process. The Li-AG/PAM DN hydrogel electrolyte possesses extremely excellent mechanical properties with a compression strength of 150 MPa, a fracture compression strain of above 99.9%, a tensile strength of 1103 kPa, and an elongation at break of 2780%, greatly superior to those have been reported. It also achieves a high ionic conductivity of 41 mS cm-1 originating from its interconnected three-dimensional porous network structure that provides a three-dimensional channel for ionic migration. Compared to the SC applying Li2SO4 aqueous solution electrolyte, the corresponding flexible Li-AG/PAM DN hydrogel electrolyte-SC presents lower charge-transfer resistance, better ionic diffusion, being closer to ideal capacitive behaviors, superior rate capability, and better cycling stability, owing to the improved ionic transport in the Li-AG/PAM DN hydrogel electrolyte and electrode interfaces. Moreover, after testing with overcharge, short circuit, and high temperature, the capacitance of the Li-AG/PAM DN hydrogel electrolyte-SC can still be well maintained. Furthermore, the electrochemical properties of the Li-AG/PAM DN hydrogel electrolyte-SC remain almost intact under different compression strains/bending angles and even after 1000 compression/bending cycles. It is expected that the Li-AG/PAM DN hydrogel electrolyte may have broad applications in modern flexible and wearable electronics.

Inward Budding and Endocytosis of Membranes Regulated by de Novo Designed Peptides.

Protein-mediated endocytosis of membrane is a key event in biological system. The mechanism, however, is still not clear. Using a de novo designed bola-type peptide KKKLLLLLLLLKKK (K3L8K3) as a protein mimic, we studied how it induced giant unilamellar vesicle (GUV) to form inward buds or endocytosis at varying conditions. Results show that the inward budding is initiated as the charged lipids are neutralized by K3L8K3, which results in a negative spontaneous curvature. If the charged lipids have unsaturated tails, the buddings are slim fibrils, which can further wrap into a spherical structure. In the case of saturated charged lipids, the buddings are rigid tubules, stable in the studied time period. The unsaturated lipid to saturated lipid ratio in the mother membrane is another key parameter governing the shape and dynamics of the buds. A complete endocytosis is observed when K3L8K3 is attached with a hydrophobic moiety, suggesting that hydrophobic interaction helps the buds to detach from the mother membrane. The molecules in the surrounding medium, such as negatively charged oligonucleotides, are engulfed into the GUV via endocytosis pathway induced by K3L8K3. Our study provides a novel strategy for illustrating the endocytosis mechanism by using peptides of simple sequence.

Hydrogen-Bonded Polymer-Small Molecule Complexes with Tunable Mechanical Properties.

A novel type of polymeric material with tunable mechanical properties is fabricated from polymers and small molecules that can form hydrogen-bonded intermolecular complexes (IMCs). In this work, poly(vinyl alcohol) (PVA)-glycerol hydrogels are first prepared, and then they are dried to form IMCs. The tensile strengths and moduli of IMCs decrease dramatically with increasing glycerol content, while the elongations increase gradually. The mechanical properties are comparable with or even superior to those of common engineering plastics and rubbers. The IMCs with high glycerol content also show excellent flexibility and cold-resistance at subzero temperatures. Cyclic tensile and stress relaxation tests prove that there is an effective energy dissipation mechanism in IMCs and dynamic mechanical analysis confirms their physical crosslinking nature. FTIR and NMR characterizations prove the existence of hydrogen bonding between glycerol and PVA chains, which suppresses the crystallization of PVA from X-ray diffraction measurement. These PVA-glycerol IMCs may find potential applications in barrier films, biomedical packaging, etc., in the future.