Bottom-up synthetic biology aims to engineer artificial cells capable of responsive behaviors by using a minimal set of molecular components. An important challenge toward this goal is the development of programmable biomaterials that can provide active spatial organization in cell-sized compartments. Here, we demonstrate the dynamic self-assembly of nucleic acid (NA) nanotubes inside water-in-oil droplets. We develop methods to encapsulate and assemble different types of DNA nanotubes from programmable DNA monomers, and demonstrate temporal control of assembly via designed pathways of RNA production and degradation. We examine the dynamic response of encapsulated nanotube assembly and disassembly with the support of statistical analysis of droplet images. Our study provides a toolkit of methods and components to build increasingly complex and functional NA materials to mimic life-like functions in synthetic cells.

Liquid–liquid phase separation (LLPS) is a common phenomenon underlying the formation of dynamic membraneless organelles in biological cells, which are emerging as major players in controlling cellular functions and health. The bottom-up synthesis of biomolecular liquid systems with simple constituents, like nucleic acids and peptides, is useful to understand LLPS in nature as well as to develop programmable means to build new amorphous materials with properties matching or surpassing those observed in natural condensates. In particular, understanding which parameters determine condensate growth kinetics is essential for the synthesis of condensates with the capacity for active, dynamic behaviors. Here we use DNA nanotechnology to study artificial liquid condensates through programmable star-shaped subunits, focusing on the effects of changing subunit size. First, we show that LLPS is achieved in a 6-fold range of subunit size. Second, we demonstrate that the rate of growth of condensate droplets scales with subunit size. Our investigation is supported by a general model that describes how coarsening and coalescence are expected to scale with subunit size under ideal assumptions. Beyond suggesting a route toward achieving control of LLPS kinetics via design of subunit size in synthetic liquids, our work suggests that particle size may be a key parameter in biological condensation processes.

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.

Phase separation of molecular condensates is emerging as a key mechanism in biology and biomaterials science. A major advantage of condensates is their capacity to form and reconfigure dynamically, generating responsive compartments that organize molecular targets and reactions in both space and time, in the absence of membranes. While condensation is known to depend on environmental conditions such as temperature and ionic strength, biological condensates in nature are likely influenced by fluctuating biochemical signals with high specificity. Here we ask whether the behavior of artificial condensates can be controlled via chemical reactions by design. Through theory and experiments we examine a model problem in which a phase separating component participates in chemical reactions that activate and deactivate its ability to self-attract. Our theoretical model indicates that such reactions have effects comparable to temperature, and illustrates the dependence of condensate kinetics on reaction parameters. We experimentally realize our model problem through a platform that combines DNA nanostar motifs to generate condensate droplets, and strand displacement reactions to kinetically control the nanostar valency. Our results show that DNA condensate dissolution and growth can be controlled reversibly via toehold-mediated strand displacement, and we characterize the influence of toehold and invasion domains, nanostar size, and nanostar valency. In some cases, the reduction of nanostar valency through invasion stabilizes the droplet size. Our results provide foundational methods for the development of dynamic nucleic acid condensates with potential applications in biomaterials science, nanofabrication, and drug delivery.