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.

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.

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.

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.

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.

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.

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.

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.

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

Unlabelled: 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.

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.

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.

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.

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.

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/NaSO systems impacts droplet uniformity and partitioning of a model solute (U15 oligoRNA).

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.

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.

Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates.

Wet-dry cycling on the early Earth is thought to have facilitated production of molecular building blocks of life, but its impact on self-assembly and compartmentalization remains largely unexplored. Here, we investigate dehydration/rehydration of complex coacervates, which are membraneless compartments formed by phase separation of polyelectrolyte solutions. Solution compositions are identified for which tenfold water loss results in maintenance, disappearance, or appearance of coacervate droplets.

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.

Particle-Based Reconfigurable Scattering Masks for Lensless Imaging.

Light scattering is typically undesired in optical systems as it often introduces defects or otherwise negatively impacts device performance. However, rather than being a hindrance, scattering can also be exploited to achieve lensless imaging using a scattering mask instead of lenses to enable devices with low-cost, compact construction, and yet a large field of view. Lensless imaging can benefit greatly from the ability to dynamically tune the scattering pattern produced by the mask; however, this often results in increased complexity and cost.

Formation of Multiphase Complex Coacervates and Partitioning of Biomolecules within them.

Biological systems employ liquid-liquid phase separation to localize macromolecules and processes. The properties of intracellular condensates that allow for multiple, distinct liquid compartments and the impact of their coexistence on phase composition and solute partitioning are not well understood. Here, we generate two and three coexisting macromolecule-rich liquid compartments by complex coacervation based on ion pairing in mixtures that contain two or three polyanions together with one, two, or three polycations.

Polyanion-Assisted Ribozyme Catalysis Inside Complex Coacervates.

Owing to their ability to encapsulate biomolecules, complex coacervates formed by associative phase separation of oppositely charged polyelectrolytes have been postulated as prebiotic nonmembranous compartments (NMCs). Recent studies show that NMCs sequester RNA and enhance ribozyme reactions, a critical tenet of the RNA World Hypothesis. As RNA is negatively charged, it is expected to interact with polycationic coacervate components.

Lipid Vesicle-Coated Complex Coacervates.

Compartmentalization by complex coacervation is important across a range of different fields including subcellular and prebiotic organization, biomedicine, food science, and personal care products. Often, lipid self-assemblies such as vesicles are also present intracellularly or in commercial formulations. A systematic understanding of how phospholipid vesicles interact with different complex coacervates could provide insight and improve control over these systems.