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...

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. Achieving 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 DNA and an azobenzene cation. The droplets disassemble and reassemble under UV and blue light, respectively, due to azobenzene trans/cis photoisomerisation. Sequestration and release of captured oligonucleotides follow the dynamics of phase separation such that light‐activated transfer, mixing, hybridization, and trafficking of the oligonucleotides can be controlled in binary populations of the droplets. Our results open perspectives for the spatiotemporal control of DNA coacervates and provide a step towards the dynamic regulation of synthetic protocells.