Polynucleotides in Cellular Mimics: Coacervates and Lipid Vesicles
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Current Opinion in Colloid & Interface Science 26 (2016) 50–57 Contents lists available at ScienceDirect Current Opinion in Colloid & Interface Science journal homepage: www.elsevier.com/locate/cocis Polynucleotides in cellular mimics: Coacervates and lipid vesicles Jeffrey R Vieregg a, T-Y Dora Tang b,⁎ a Institute for Molecular Engineering, University of Chicago, 5640 South Ellis Avenue, Chicago, IL 60637, USA b Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany article info abstract Article history: In this review, we examine the interaction of nucleic acids with cell-like structures based on liquid–liquid phase Received 10 June 2016 separation of charged molecules (complex coacervation) and amphiphilic self-assembly (lipid vesicles). We Received in revised form 22 August 2016 discuss the mechanisms of their assembly and describe how they can be used as models for origin of life studies Accepted 2 September 2016 and for understanding two recently-described phenomena in modern cells: membrane-free organelles and Available online 28 September 2016 exosomes. Hybrid cells with increased structural complexity are highlighted and we then briefly explore how strategies based on electrostatic and hydrophobic assembly can be used for designing and synthesizing delivery Keywords: Coacervate agents for therapeutic nucleic acids. While the physical mechanisms of self-assembly vary, both strategies Lipid vesicle provide viable routes for generating minimal compartmentalized systems, modeling cellular pathways, and for Self-assembly rational design of new synthetic cells for technological applications. Protocells © 2016 Elsevier Ltd. All rights reserved. Nucleic acids Drug delivery Therapeutics Synthetic cells 1. Introduction provide localization for heterogeneous reactions and are essential for the electrochemical gradients used for essential modern processes such Cells are the basic unit of self-replicating life on Earth. However, as ATP synthesis, but can require complex protein machinery to create, the complete mechanistic pathways that drive replication, homeostasis, maintain, and regulate flows of chemicals that cannot pass through them. information propagation and evolution, are still poorly understood. In Modern cellular biochemistry also provides clues with respect to addition, how biological life might have evolved from simple chemical information propagation in early life scenarios. Of the various biological processes remains an open, unanswered question. Therefore, synthesiz- molecules, only nucleic acids (RNA and DNA) have the capability to ing abiotic cellular analogs, or “protocells”, is an important route for catalyze chemical reactions and template their own genetic propaga- describing and understanding biological mechanisms and offers in- tion. The “RNA World” hypothesis imagines a scenario on prebiotic triguing models to describe evolutionary pathways in the pre-biotic earth where self-replicating RNA molecules were both enzymes and world [1,2]. In order to synthesize suitable protocells it is important genes; today, these functionalities are primarily fulfilled by proteins to connect molecular structure with self-assembly processes capable and DNA. As there is no material evidence for the “RNA World” hypoth- of supporting key features required for life, including concentration of esis it remains speculative [6], however the discovery that ribosomes functional molecules, creation of distinct chemical environments, and are, at their core, ribozymes [7•], strongly indicates that RNA preceded information propagation. proteins in the chemical evolution of early life, thus highlighting the Compartmentalization provides a mechanism for increasing local importance of considering polynucleotides in early life scenarios and concentrations of enzymes and substrates sufficiently to drive chemical in modern natural and synthetic self-assembled structures. reactions, and is a necessary feature for enriching functional molecules In this review, we discuss the interaction of nucleic acids with two and their precursors. One plausible scenario for prebiotic compartmental- types of self-assembled systems that display cell-like properties: complex ization describes the spontaneous assembly and concomitant chemical coacervates and hydrophobic assemblies such as micelles and vesicles. enrichment of charged molecules to form membrane free droplets called For each type, we briefly discuss their mechanisms of formation and coacervates [3••]. An alternative route to chemical compartmentalization how these processes may be applied to early life scenarios, compartmen- is via the formation of lipid membrane bound compartments, analogous talization phenomena in modern biology, and for therapeutics. All of to the membrane structure of modern cells [1,4,5]. Membranes also these topics are exciting areas of research in their own right, and space does not permit a full exploration of any of them here. We instead attempt to highlight recent research results and interesting connections, ⁎ Corresponding author. while referring the reader to reviews for more comprehensive explana- E-mail address: [email protected] (T.-Y.D. Tang). tions of the underlying phenomena. http://dx.doi.org/10.1016/j.cocis.2016.09.004 1359-0294/© 2016 Elsevier Ltd. All rights reserved. J.R. Vieregg, T.-Y.D. Tang / Current Opinion in Colloid & Interface Science 26 (2016) 50–57 51 2. Electrostatic assembly: complex coacervate protocells form from small molecular weight molecules, particularly nucleotides and their activated derivatives. This was first demonstrated in 2011 by Interactions between oppositely charged molecules can lead to Koga et al. [22••], who showed that coacervate microdroplets (Fig. 2a) phase separation, forming either liquid droplets (complex coacervation) could be formed from nucleoside triphosphates (ATP), diphosphates or solid precipitates depending on the length, charge density and type (ADP, FAD, NAD), and monophosphates (AMP) when mixed with of macromolecules. Complex coacervation refers to spontaneous forma- short (2–10 amino acid (aa)) lysine polypeptides (OLys) that might tion of a polymer-rich liquid phase in dynamic equilibrium with a plausibly be produced by prebiotic processes [23]. This study showed polymer-poor phase [8,9••] via electrostatic interactions between oppo- that phase separation of small molecular weight ions has many similari- sitely charged molecules in aqueous solution. This process was first ties to complexation of larger polyelectrolytes. The dependence on elec- described by Bungenberg de Jong in the 1920s in mixtures of gelatin trostatic interactions, for example, is shown by the increase of the critical (polycation) and gum arabic (polyanion) [10]. While a complete quanti- concentration required for coacervation (CCC) with decreasing negative tative model is still lacking, many aspects of complex coacervation are charge from ATP N ADP N AMP (Fig. 2b). In addition, they found that qualitatively understood for long polyelectrolytes. The formation of increasing the molecular weight of Poly(diallyldimethylammonium) macroion pairs between oppositely charged molecules is driven by an (Poly(DADMAC)) from 150 to 275 kDa increased charge neutralization increase of entropy from the release of low valence counterions and (with ATP) from 70 to 90%, These results suggest that increased hydro- water rearrangement as they associate. The macroion pairs then assem- phobicity, decreased solubility and increased orientational freedom ble into larger clusters, which leads to macroscopic phase separation from longer polymer chains all contribute to increasing charge neutrali- (Fig. 1a) [9••,11]. Significantly, the dense phase, while highly enriched zation at the CCC. in polymer, remains highly hydrated with a large concentration of Nucleotide coacervate droplets exhibit dynamic behaviors such as counterions. The relative importance of ion pairing, hydration effects, coalescence and surface wetting (Fig. 2a) but remain stable over a and long-range electrostatics remains uncertain [12–14••] as do the broad pH and temperature range (between pH 2 and 10 and up to factors determining whether a particular macroion pair will form a 80 °C respectively). The coacervates also produce substantial chemical liquid or a solid condensed complex [15]. As polyelectrolytes can be enrichment of charged molecules, with OLys (2–10 aa)/ATP droplets chemically diverse the process of complex coacervation is molecularly displaying a twenty-fold increase in ATP concentration relative to the non-specific. Indeed, since the 1920s complex coacervation has been dilute phase [22] and 15 kD poly(allylamine) droplets providing a observed between hundreds of different natural and synthetic polymers 300× increase of fluorescent solutes, as well as substantial enrichment [8,16,17••]. Their properties including ease of formation, high viscosity, of Mg2+ and RNA oligonucleotides [24]. The dielectric constant of the strong adhesion and high encapsulation efficiencies have led to a droplet interior is lower than that of the dilute phase, which provides range of industrial applications including food additives [18] and elec- a mechanism for sequestering low-charge molecules such as protein tronic ink [19], as well as therapeutic assemblies (see Section 5). enzymes. Sequestration provides a molecular route to accelerate bio- In 1924, Alexander Oparin put forward the idea that colloidal chemical reactions, primitive metabolism, and information