High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret To cite this version: Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret. High- Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics. ChemSystem- sChem, Wiley, 2020, 10.1002/syst.202000022. hal-02870965 HAL Id: hal-02870965 https://hal.archives-ouvertes.fr/hal-02870965 Submitted on 17 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Articles ChemSystemsChem doi.org/10.1002/syst.202000022 1 2 3 High-Throughput Synthesis and Screening of Functional 4 5 Coacervates Using Microfluidics 6 + [a] + [b, d] [a] [b, d] 7 Thomas Beneyton , Celina Love , Mathias Girault, T.-Y. Dora Tang,* and [a, c] 8 Jean-Christophe Baret* 9 10 11 To understand how membrane-free subcompartmentalization reaction rates of individual subcompartments and their sur- 12 can modulate biochemical reactions by coupled spatial enzyme rounding aqueous environment at the single coacervate level. 13 localization with substrate and product partitioning, we use Our results show that sub-compartmentalisation increases the 14 microfluidic strategies to synthesize, stabilize and characterize overall rates of reactions. This bottom-up synthetic strategy for 15 micron-sized functional coacervates in waterÀ oil emulsions. Our the production of synthetic organelles offers a physical model 16 methodologies have allowed for the first time to quantitatively for membrane-free compartmentalization in biology and pro- 17 characterize partition coefficients of a broad range of different vides insights into the role of sub-compartmentalisation in 18 molecules with different coacervate chemistries and to measure regulating out-of-equilibrium behaviours in biological systems. 19 20 1. Introduction tion is based on membrane bound compartments such as the 21 mitochondria, nucleus and Golgi apparatus or on membrane- 22 Compartmentalization is an essential feature of life, where free droplets such as stress granules or P-granules which are 23 hierarchical assembly of sub compartments provides a way to collectively known as condensates. The latter are believed to 24 spatially organize biology. Cellular micro and nano-compart- form via a liquid-liquid phase separation process of intrinsically 25 ments allows distinct chemical environments to coexist and disordered proteins (IDPs), RNA and structured proteins[10,11] and 26 support multi-step reactions. In addition, compartmentalization are hypothesized to regulate biological function by regulating 27 supports the generation and sustenance of chemical gradients the sequestration and therefore the local concentration of 28 essential to maintain out-of-equilibrium processes in cells.[1] molecules. Whilst these condensates have been hypothesized 29 Oparin hypothesized that compartmentalization would have to have potential implications in disease, gene expression 30 been crucial at the onset of life to enable the organization of regulation[12] and signaling,[13,14] how these membrane free 31 chemical and biochemical reactions by providing chemical droplets may alter out-of equilibrium behaviours is still not well 32 centers in which selected molecules were concentrated for understood. It can often be difficult to characterize the 33 prebiotic chemistry and/or biology.[2–9] In modern biology it is sequestration of different molecules into biological condensates 34 well known that compartmentalization occurs across length in vivo due to the molecular complexity and in vitro due to the 35 scales, from the sub-cellular level to the macroscopic assembly difficulty in expressing and purifying condensate forming 36 of compartments in multicellular organisms. Compartmentaliza- proteins. 37 Coacervate microdroplets formed through liquid-liquid 38 + phase separation between oppositely charged polyelectrolytes 39 [a] Dr. T. Beneyton, Dr. M. Girault, Prof. J.-C. Baret Univ. Bordeaux, CNRS and small molecules offer an alternative synthetic model to 40 CRPP, UMR 5031 biological condensates as studies have shown that the mecha- 41 115 avenue Albert Schweitzer nism of biological condensate formation is partly driven by 42 33600 Pessac (France) E-mail: [email protected] coacervation.[15,16] In addition, these chemically enriched mem- 43 + [b] C. Love, Dr. T.-Y. D. Tang brane-free droplets with a highly charged and crowded 44 Max Planck Institute for Molecular Cell Biology and Genetics interior[17,18] passively upconcentrate molecules and have been 45 Pfotenhauerstrasse 108 01307 Dresden (Germany) shown to support enzymatic reactions where alterations in 46 E-mail: [email protected] diffusion length scales or in secondary structure of enzymes 47 [c] Prof. J.-C. Baret affects the rates of reactions[19–24] thereby modulating biochem- 48 Institut Universitaire de France 75005 Paris France [d] C. Love,+ Dr. T.-Y. D. Tang ical processes. Artificial systems formed by coacervation will 49 Cluster of Excellence Physics of Life therefore be interesting models to test hypothesis on the 50 TU Dresden, 01062 Dresden (Germany) dynamic properties of membrane-free organelles and offer new 51 [+] These authors contributed equally to this work routes to generate controlled mimics of those. 52 Supporting information for this article is available on the WWW under Moreover, coacervates represent interesting materials[25] and 53 https://doi.org/10.1002/syst.202000022 © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This are promising tools in bottom-up synthetic biology, where one 54 is an open access article under the terms of the Creative Commons Attri- of its aims is to construct artificial living systems from 55 bution Non-Commercial License, which permits use, distribution and re- elementary ingredients and modules.[26] Despite their advan- 56 production in any medium, provided the original work is properly cited and is not used for commercial purposes. tages for a range of applications the assembly of functional 57 ChemSystemsChem 2020, 2, e2000022 (1 of 9) © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Wiley VCH Dienstag, 26.05.2020 2099 / 164441 [S. 1/9] 1 Articles ChemSystemsChem doi.org/10.1002/syst.202000022 coacervate-based compartments is hindered by the intrinsic underlying mechanism of the transport process. We provide 1 transient nature of the structures and the difficulty in handling quantitative measurements with statistics over large popula- 2 and characterizing known concentrations of molecules within tions of coacervates. Our quantitative approach demonstrates 3 the coacervates at the micron scale. Recently, the control of for the first time that transport in coacervates at the micron 4 coacervate formation was achieved in micron-sized compart- scale is driven by equilibrium processes, without energy barriers 5 ments using microfluidics,[27,28] with the demonstration that the to partitioning. 6 coacervation process can be controlled by a chemical reaction 7 acting on the molecule that makes up the coacervate. Whilst 8 such a synthetic biology approach represents progress in 2. Results and Discussion 9 building structural representations of compartmentalization 10 there still remains key challenges for the synthesis and To achieve stable individuated coacervate microdroplets we 11 characterisation of functional model systems that can be used microfluidics to spontaneously assemble coacervates with- 12 produced reliably and robustly in a high throughput manner. in water-in-oil (w/o) droplets. Traditionally, coacervate micro- 13 New microfluidic methods for the high-throughput produc- droplets are formed by mixing stock aqueous solutions, by 14 tion, functionalisation and analysis of coacervates would hand, containing either a polycation (Polylysine (Plys) or poly 15 provide unprecedented quantitative approaches of molecular (diallyldimethylammounium chloride) (PDDA)) with a polyanion 16 dynamic processes on the single droplets level at high (carboxymethylÀ dextran (CMÀ Dex) or adenosine triphosphate 17 throughput and provide rational models for enzymatically (ATP)) at fixed molar ratios (Figure S1). When prepared by hand, 18 active subcompartments as found in biological systems.[29,30] coacervates were highly polydisperse as observed by optical 19 To address these issues, we have designed and synthesised microscopy (Figure S2), where diameters typically span one 20 an active membrane-free subcompartment with coupled fea- order of magnitude (and therefore volumes vary over three 21 tures of localized enzyme reactions and passive molecular orders of magnitude). We observe that the droplets coarsen 22 sequestration. Structurally, this comprises a coacervate micro- with a continuous change of coacervate size distribution over 23 droplet surrounded by a finite volume of aqueous media timescales ranging from hours to days. Eventually, all of the 24 provided by a surfactant stabilized water-in oil