Hybrid Protocells Based on Coacervate-Templated Fatty Acid Vesicles Combine Improved Membrane Stability with Functional Interior Protocytoplasm.

Prebiotically-plausible compartmentalization mechanisms include membrane vesicles formed by amphiphile self-assembly and coacervate droplets formed by liquid-liquid phase separation. Both types of structures form spontaneously and can be related to cellular compartmentalization motifs in today's living cells. As prebiotic compartments, they have complementary capabilities, with coacervates offering excellent solute accumulation and membranes providing superior boundaries. Herein, protocell models constructed by spontaneous encapsulation of coacervate droplets by mixed fatty acid/phospholipid and by purely fatty acid membranes are described. Coacervate-supported membranes form over a range of coacervate and lipid compositions, with membrane properties impacted by charge-charge interactions between coacervates and membranes. Vesicles formed by coacervate-templated membrane assembly exhibit profoundly different permeability than traditional fatty acid or blended fatty acid/phospholipid membranes without a coacervate interior, particularly in the presence of magnesium ions (Mg2+). While fatty acid and blended membrane vesicles are disrupted by the addition of Mg2+, the corresponding coacervate-supported membranes remain intact and impermeable to externally-added solutes. With the more robust membrane, fluorescein diacetate (FDA) hydrolysis, which is commonly used for cell viability assays, can be performed inside the protocell model due to the simple diffusion of FDA and then following with the coacervate-mediated abiotic hydrolysis to fluorescein.

Cell-Free Expressed Membraneless Organelles Inhibit Translation in Synthetic Cells.

Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.

Kinetic control of shape deformations and membrane phase separation inside giant vesicles.

A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.

Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems.

Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.

Structure-seq of tRNAs and other short RNAs in droplets and in vivo.

There is a multitude of small (<100nt) RNAs that serve diverse functional roles in biology. Key amongst these is transfer RNA (tRNA), which is among the most ancient RNAs and is part of the translational apparatus in every domain of life. Transfer RNAs are also the most heavily modified class of RNAs. They are essential and their misregulation, due to mutated sequences or loss of modification, can lead to disease. Because of the severe phenotypes associated with mitochondrial tRNA defects in particular, the desire to deliver repaired tRNAs via droplets such as lipid nanoparticles or other compartments is an active area of research. Here we describe how to use our tRNA Structure-seq method to study tRNAs and other small RNAs in two different biologically relevant contexts, peptide-rich droplets and in vivo.

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life.

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Here, we detail next-generation sequencing (NGS) experiments performed in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Notably, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.

Effect of Polypeptide Complex Coacervate Microenvironment on Protonation of a Guest Molecule.

Complex coacervate droplets formed by the liquid-liquid phase separation of polyelectrolyte solutions capture several important features of membraneless organelles including their ability to accumulate guest molecules and to provide distinct microenvironments. Here, we examine how polyions in complex coacervates can influence localized guest molecules, leading to a shifted protonation state of the guest molecule in response to its electrostatic environment. A fluorescent ratiometric pH indicator dye was used as a model guest molecule able to report its protonation state in the coacervate phase. Experimentally observed differences in dye-reported local apparent pH inside versus outside of coacervate droplets were largest for polyion pairs having lower salt stabilities and/or larger polyion length mismatch, which we attribute to the relative concentration of open sites on polyions within the coacervates based on theoretical calculations. Using the transfer matrix method, we confirmed that theoretical phase diagrams and critical salt stabilities generated for each polyion pair were consistent with experimental turbidity measurements and estimated the amount of available binding sites on polyions for guest molecules. We conclude that dye molecules likely experience an effective pKa shift due to interactions with coacervate polyions rather than reporting directly on local proton concentrations. Such a local pKa shift can also be anticipated for other guest molecules having protonatable groups, including, for example, many metabolites, ligands, and/or drug molecules that partition into coacervates or membraneless organelles based on ion pairing interactions.

Cell-free expressed membraneless organelles sequester RNA in synthetic cells.

Compartments within living cells create specialized microenvironments, allowing for multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation, such as P-granules and nucleoli. Synthetic minimal cells have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

RNA folding studies inside peptide-rich droplets reveal roles of modified nucleosides at the origin of life.

Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life. One Sentence Summary: We demonstrate that RNA folds into native secondary and tertiary structures in protocell models and that this is favored by covalent modifications, which is critical for the origins of life.

Phase-specific RNA accumulation and duplex thermodynamics in multiphase coacervate models for membraneless organelles.

Liquid-liquid phase separation has emerged as an important means of intracellular RNA compartmentalization. Some membraneless organelles host two or more compartments serving different putative biochemical roles. The mechanisms for, and functional consequences of, this subcompartmentalization are not yet well understood. Here we show that adjacent phases of decapeptide-based multiphase model membraneless organelles differ markedly in their interactions with RNA. Single- and double-stranded RNAs preferentially accumulate in different phases within the same droplet, and one phase is more destabilizing for RNA duplexes than the other. Single-phase peptide droplets did not capture this behaviour. Phase coexistence introduces new thermodynamic equilibria that alter RNA duplex stability and RNA sorting by hybridization state. These effects require neither biospecific RNA-binding sites nor full-length proteins. As such, they are more general and point to primitive versions of mechanisms operating in extant biology that could aid understanding and enable the design of functional artificial membraneless organelles.

Microfluidic Control of Coexisting Chemical Microenvironments within Multiphase Water-in-Fluorocarbon Droplets.

The use of aqueous polymer-based phase separation within water-in-oil emulsion droplets provides a powerful platform for exploring the impact of compartmentalization and preferential partitioning on biologically relevant solutes. By forming an emulsion, a bulk solution is converted into a large number of chemically isolated microscale droplets. Microfluidic techniques provide an additional level of control over the formation of such systems. This enables the selective production of multiphase droplets with desired solution compositions and specific characteristics, such as solute partitioning. Here, we demonstrate control over the chemical microenvironment by adjusting the composition to increase tie line length for poly(ethylene glycol) (PEG)-dextran aqueous two-phase systems (ATPS) encapsulated within multiphase water-in-fluorocarbon oil emulsion droplets. Through rational adjustment of microfluidic parameters alone, ATPS droplets containing differing compositions could be produced during the course of a single experiment, with the produced droplets demonstrating a controllable range of tie line lengths. This provided control over partitioning behavior for biologically relevant macromolecules such that the difference in local protein concentration between adjacent phases could be rationally tuned. This work illustrates a broadly applicable technique to rationally create emulsified multiphase aqueous systems of desired compositions through the adjustment of microfluidic parameters alone, allowing for easy and rapid screening of various chemical microenvironments.

RNA sequence and structure control assembly and function of RNA condensates.

Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.

Phospholipid Membrane Formation Templated by Coacervate Droplets.

We report the formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated by what appear to be continuous lipid bilayers. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Within the same population, in addition to coacervate droplets having intact lipid membrane coatings, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and some are coated with multilayered membranes. Membranes surrounding protein-based coacervate droplets provided protection from a protease added to the external solution. The simplicity of producing artificial cells having a coacervate model cytoplasm surrounded by a model membrane is at the same time interesting as a potential mechanism for prebiotic protocell formation and appealing for biotechnology. We anticipate that such structures could serve as a new type of model system for understanding interactions between intracellular phases and cell or organelle membranes, which are implicated in a growing number of processes ranging from neurotransmission to signaling.

Formation and properties of liposome-stabilized all-aqueous emulsions based on PEG/dextran, PEG/Ficoll, and PEG/sulfate aqueous biphasic systems.

Vesicle-stabilized all-aqueous emulsion droplets are appealing as bioreactors because they provide uniform encapsulation via equilibrium partitioning without restricting diffusion in and out of the interior. These properties rely on the composition of the aqueous two-phase system (ATPS) chosen for the emulsion and the structure of the interfacial liposome layer, respectively. Here, we explore how changing the aqueous two-phase system from a standard poly(ethyleneglycol), PEG, 8 kDa/dextran 10 kDa ATPS to PEG 8 kDa/Ficoll 70 kDa or PEG 8 kDa/Na2SO4 systems impacts droplet uniformity and partitioning of a model solute (U15 oligoRNA). We also compare liposomes formed by two different methods, both of which begin with multilamellar, polydisperse vesicles formed by gentle hydration: (1) extrusion, which produced vesicles of 150 nm average diameter, and (2) vortexing, which produced vesicles of 270 nm average diameter. Our data illustrate that while droplet uniformity and stability are somewhat better for samples based on extruded vesicles, extrusion is not necessary to create functional microreactors, as emulsions stabilized with vortexed liposomes are just as effective at solute partitioning and allow diffusion across the droplet's liposome corona. This work expands the compositions possible for liposome-stabilized, all-aqueous emulsion droplet bioreactors, making them amenable to a wider range of potential reactions. Replacing the liposome extrusion step with vortexing can reduce time and cost of bioreactor production with only modest reductions in emulsion quality.

Practical considerations for generation of multi-compartment complex coacervates.

We discuss preparation of experimental models for multi-compartment membraneless organelles in which distinct compositions are maintained indefinitely for macromolecule-rich phases in contact with each other. These model systems are based on the physical chemistry phenomenon of complex coacervation. In complex coacervation, liquid-liquid phase separation occurs due to ion pairing interactions between oppositely charged polyelectrolytes. This mechanism can drive the associative phase separation of proteins and nucleic acids, the major macromolecular components of membraneless organelles. Here we provide examples, advice and practical considerations for the design, generation, and analysis of multi-compartment complex coacervates. These structures are of interest to compartmentalize the interior of artificial cells and as models for the intracellular membraneless organelles of biological cells.

Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments.

Multivalent polyions can undergo complex coacervation, producing membraneless compartments that accumulate ribozymes and enhance catalysis, and offering a mechanism for functional prebiotic compartmentalization in the origins of life. Here, we evaluate the impact of lower, more prebiotically-relevant, polyion multivalency on the functional performance of coacervates as compartments. Positively and negatively charged homopeptides with 1-100 residues and adenosine mono-, di-, and triphosphate nucleotides are used as model polyions. Polycation/polyanion pairs are tested for coacervation, and resulting membraneless compartments are analyzed for salt resistance, ability to provide a distinct internal microenvironment (apparent local pH, RNA partitioning), and effect on RNA structure formation. We find that coacervates formed by phase separation of the shorter polyions more effectively generated distinct pH microenvironments, accumulated RNA, and preserved duplexes than those formed by longer polyions. Hence, coacervates formed by reduced multivalency polyions are not only viable as functional compartments for prebiotic chemistries, they can outperform higher molecular weight analogues.

Linear and nonlinear chiroptical response from individual 3D printed plasmonic and dielectric micro-helices.

Sub-wavelength chiral resonators formed from artificial structures exhibit exceedingly large chiroptical responses compared to those observed in natural media. Owing to resonant excitation, chiral near fields can be significantly enhanced for these resonators, holding great promise for developing enantioselective photonic components such as biochemical sensors based on circular dichroism (CD) and spin-dependent nonlinear imaging. In the present work, strong linear and nonlinear chiroptical responses (scattering CD > 0.15 and nonlinear differential CDs > 0.4) at visible and near infrared frequencies are reported for the first time for individual micrometer-scale plasmonic and dielectric helical structures. By leveraging dark-field spectroscopy and nonlinear optical microscopy, the circular-polarization-selective scattering behavior and nonlinear optical responses (e.g., second harmonic generation and two-photon photoluminescence) of 3D printed micro-helices with feature sizes comparable to the wavelength (total length is ∼5λ) are demonstrated. These micro-helices provide potential for readily accessible photonic platforms, facilitating an enantiomeric analysis of chiral materials. One such example is the opportunity to explore ultracompact photonic devices based on single, complex meta-atoms enabled by state-of-the-art 3D fabrication techniques.