Photoswitchable Phase Separation and Oligonucleotide Trafficking in DNA Coacervate Microdroplets

Abstract

Coacervate microdroplets produced by liquid-liquid phase separation have been used as synthetic protocells that mimic the dynamical organization of membrane-free organelles in living systems.A chieving spatiotemporal control over droplet condensation and disassembly remains challenging. Herein, we describe the formation and photoswitchable behavior of light-responsive coacervate droplets prepared from mixtures of double-stranded DNAa nd an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively,d ue to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides followt he dynamics of phase separation such that light-activated transfer,m ixing,h ybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets.O ur results open perspectives for the spatiotemporal control of DNAc oacervates and provide as tep towards the dynamic regulation of synthetic protocells.The dynamic compartmentalization of biological components in space and time is ah allmark of functional synchronization in living cells.Biomolecular condensates formed via liquid-liquid phase separation are dynamic subcellular compartments that are actively formed and dissolved in response to environmental cues. [1,2] They play acrucial role for example in the compaction of polynucleotides and regulation of genetic expression. [3,4] Microdroplets produced in vitro by associative (coacervates) or segregative (aqueous two-phase systems) liquid-liquid phase separation have recently been used as synthetic models of dynamic protocells. [5][6][7] These membrane-free molecularly crowded compartments seques-ter functional biomolecules [5,8] and support enzymatic activity, [9,10] protein folding, [11] RNAc atalysis, [12,13] and cell-free protein expression. [14] Polynucleotides have been used as scaffold components to assemble coacervate droplets, [15][16][17][18][19] and selective sequestration of guest polynucleotides has been demonstrated in preformed coacervates. [12,20,21] Owing to their liquid-like nature and the weak molecular interactions,s ynthetic coacervate droplets can dynamically respond to external stimuli. [7,22,23] Fori nstance,t heir formation and dissolution has been achieved by changes in pH, [5] temperature, [24][25][26] ionic strength, [27,28] and more recently by enzymemediated catalytic activity. [15,[29][30][31][32] However,p latforms enabling the rapid and localized actuation of polynucleotide microphase separation have not yet been developed. Due to its favourable spectral and spatiotemporal capabilities,l ight holds great promise as aprogrammable trigger for controlling the reversible assembly and disassembly of coacervate droplets.L ight has been used to control synthetic microcompartments, [33][34][35] trigger biomolecular condensation in cellulo via optogenetic tools, [36,37] promote the dynamical shaping of soft colloids, [38] and induce the reversible compaction of single DNAc hains. [39][40][41] Here,w ee xploit t...

Response‐Retaliation Behavior in Synthetic Protocell Communities

“…Dispersed communities of synthetic protocells based on mixed populations of lipid vesicles, polymersomes, colloidosomes, proteinosomes, and coacervate microdroplets provide an attractive opportunity to develop functionally interactive microcompartmentalized systems capable of chemical communication, sensing, signal‐induced differentiation, distributed computing, oligonucleotide trafficking, and enzyme‐powered buoyancy . Increasing the complexity of these consortia by exploiting matter and energy fluxes to drive cognate interactions remains a key challenge and a critical step towards synthetic protocell ecosystems exhibiting higher order contact‐dependent behavior, such as artificial phagocytosis and prototissue assembly …”

Reversible pH‐Responsive Coacervate Formation in Lipid Vesicles Activates Dormant Enzymatic Reactions

“…This coacervation process has attracted a lot of attention as it has recently been demonstrated to be one of the driving forces of cellular condensate formation . Cytoplasmic phase de‐mixing and synthetic liquid droplets have been shown to respond reversibly to changes in pH, salt, enzymes, and light in vivo and in vitro. Here, regulation of the molecular charge or chemical structure of the coacervate‐forming components leads to the mixing and de‐mixing of the microdroplet resulting in dramatic changes in the local environment.…”

Biological phase separation: cell biology meets biophysics

“…However, such phase separation also depends on other factors including temperature, pH, salt concentration, and the length and concentration of the "coacervating" analytes (Priftis et al 2013). Upon further agitation, potentially caused by primitive geological processes such as wind, tides, or turbulence driven by fumaroles or other pressurized hydrothermal structures (Chiodini et al 2012), the separated phases In vitro-produced coacervates can segregate and compartmentalize important functional biomolecules (retaining their function and/or structure) such as DNA (Martin et al 2019), RNA (Poudyal et al 2018(Poudyal et al , 2019Drobot et al 2018), and proteins (Martin et al 2016) and can scaffold the assembly of lipid layers (Tang et al 2014). Coacervate droplets could be more permeable to small nutrients compared with fatty acid membrane vesicles and can be formed from fairly simple prebiotic components such as mononucleotides and short peptides, which potentially eliminates the need for abiotic fatty acid synthesis in primitive systems (Frankel et al 2016).…”