Liquid-liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length-dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.

This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and discuss potential roles in template-mediated non-enzymatic polymerization of RNA and other related biomolecules, functions of ribozymes and aptamers, and benefits or penalties imparted by liquid demixing. We conclude that tiny liquid droplets may have concentrated precious biomolecules and acted as bioreactors in the RNA World.

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.

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 selfassemblies 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. In this manuscript, anionic phospholipid vesicles were added to a series of different complex coacervate samples in which coacervates were formed by mixing one of five polycations with one of three (poly)anions that varied in chemical structure and length. Vesicles were found to assemble at the coacervate/continuous phase interface and/or form aggregates. We report how factors such as the charge density of polyelectrolytes and the charge ratio of cationic-to-anionic moieties impact the vesicle distribution in coacervate samples. Our findings emphasize the importance of interactions between vesicles and polycations in the dilute supernatant phase for determining whether the vesicles aggregate prior to assembly at the liquid−liquid interface. The uptake of an RNA oligonucleotide (A 15 ) was also investigated to understand the effect of these liposome coatings on diffusion into coacervate droplets. Systems in which uniform vesicle coronas assemble around coacervate droplets without restricting the entry of biomolecules such as RNAs could be of interest as bioreactors.

Enhanced spatiotemporal selectivity in photonic sensitization of dissolved molecular oxygen is an important target for improving the potential and the practical applications of photodynamic therapy. Considering the high intracellular glutathione concentrations within cancer cells, a series of BODIPY-based sensitizers that can generate cytotoxic singlet oxygen only after glutathione-mediated cleavage of the electron-sink module were designed and synthesized. Cell culture studies not only validate our design, but also suggest an additional role for the relatively hydrophobic quencher module in the internalization of the photosensitizer.

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.

Natural clay particles have been hypothesized as catalysts on the early Earth, potentially facilitating the formation of early organic (bio) molecules. Association of clay particles with droplets formed by liquidliquid phase separation could provide a physical mechanism for compartmentalization of inorganic catalysts in primitive protocells. Here we explore the distribution of natural clay mineral particles in poly(ethylene glycol) (PEG)/dextran (Dx) aqueous two-phase systems (ATPS). We compared the three main types of natural clay: kaolinite, montmorillonite and illite, all of which are aluminosilicates of similar composition and surface charge. The three clay types differ in particle size, crystal structure, and their accumulation at the ATPS interface and ability to stabilize droplets against coalescence. Illite and kaolinite accumulated at the aqueous/aqueous interface, stabilizing droplets against coalescence but not preventing their eventual sedimentation due to the mass of adsorbed particles. The ability of each clay-containing ATPS to catalyze reaction of o-phenylenediamine with peroxide to form 2,3-diaminophenazone was evaluated. We observed modest rate increases for this reaction in the presence of clay-containing ATPS over clay in buffer alone, with illite outperforming the other clays. These findings are encouraging because they support the potential of combining catalytic mineral particles with aqueous microcompartments to form primitive microreactors.Clay minerals, which are composed of aluminosilicates with layered structures, are major components of soils and sedimentary rocks and among the most abundant minerals at the surface of the Earth 1 . Clays have found a wide variety of applications since ancient times including ceramics 2 , electrochemistry 3 , organoclay/polymer nanocomposites 4 and as catalysts in chemical reactions [5][6][7] . The availability of clay minerals and their potential for catalytic activity led to the proposal of these materials as inorganic catalysts for chemical evolution of biomolecules on the early Earth 8 . Since then clay particles have been demonstrated as catalysts for polymerization of activated nucleotides and amino acids 9 , lipid self-organization 10-12 and many other possible prebiotic reactions 11,13,14 .An important step in the transition from nonliving towards living matter is thought to be compartmentalization of molecules and of reactions 15,16 . Candidates for early-Earth compartments include crevices or pores in rocks 17 , self-assemblies of lipids or simpler amphiphiles to form vesicles [18][19][20][21][22][23] , and aqueous droplets formed by liquid-liquid phase separation 15,21,24 . Combining catalytic mineral surfaces with compartments such as amphiphile vesicles or droplets formed by aqueous/aqueous phase coexistence is therefore appealing as primitive protocell models. Szostak and coworkers reported that clay minerals accelerate the spontaneous conversion of fatty acid micelles into vesicles and some clay might get encapsulated within the vesicle...

A series of pH and GSH responsive photosensitizers were designed and synthesized. pK a values were optimized by adjusting the inductive contribution of substituents to reach a pH range (6.0-7.4) relevant to the tumour microenvironment. pH-Activatable behaviour and redox mediated release of the quencher from the PS by GSH allow the construction of an AND logic operator for selective photodynamic action in aqueous solutions.The research in molecular logic gates, which was initiated by the seminal work by de Silva, 1 blossomed in the two decades that followed. 2 In addition, the limitations and the potential of this approach has become more clear. A particularly promising application of molecular logic gates may be in the field of information processing therapeutic agents. Incorporation of Boolean logic ideas in the function of therapeutic agents would be very valuable, if the same results cannot be achieved by random optimization studies. Previously, our group and others provided the early examples of the work in that direction. 3 Our first proof of principle work which linked photodynamic sensitization of a Bodipy based photosensitizer (PS) to the concentrations of sodium ions and the acidity was essentially an AND logic gate, but the system required organic solvents and organic acid to function in the desired manner. While it was considered to be noteworthy for that approach to have practical potential, an AND logic gate based enhanced selectivity should be related to cancer related biological parameters, which can generate significant changes in the photophysical character of the sensitizer in aqueous solutions.In this work, we took advantage of two characteristics of the tumour microenvironment, lower pH and higher glutathione concentrations. 4 The difference in pH between cancer tissue and healthy tissue is an easily accessible parameter for use in therapeutic activation. A number of pH-responsive polymeric materials, photosensitizers, and nanocarriers were studied to control drug release or activation. 5 However, extracellular pH of tumor cells drops to a value not below 6.0. 6 Thus, it is challenging to find a smart therapeutic system responsive to pH within the narrow near neutral range and essentially become active at around pH 6.0-6.5 and stay inactive above pH 7.0. Apart from some, 7 most related studies in the literature depend on activation at pH below 5.5, which actually requires nonselective lysosomal activation. 8 In this work, the properties of the PS are optimized for pH activatability by causing rational chemical modification on the pH responsive moiety with electron donating or withdrawing groups to adjust the pK a to the desired near-neutral value and to get enough spectral shift in acidic aqueous solutions such that protonated PSs are exclusively excited species under the conditions of interest. Thus, the overall design (Scheme 1) involves a pH responsive unit, linked to a quencher, which could be cleaved at elevated GSH concentrations. Previously, GSH has been used as a PS activator mostly t...