Sequence-Controlled Adhesion and Microemulsification in a Two-Phase System of DNA Liquid Droplets

Abstract

Membrane-less organelles, the liquid droplets formed via liquid−liquid phase separation (LLPS) of biomolecules in cells, act to organize intracellular components into multiple compartments. As a model for this process, and as a potential vehicle for in vitro exploitation of its properties, we explore here a synthetic multiphase LLPS system consisting of a mixture of selfassembled DNA particles. The particles, termed "DNA nanostars" (NSs), consist of four double-stranded DNA arms that each terminate in a single-stranded overhang. NSs condense into droplets due to overhang hybridization. Using two types of NSs with orthogonal overhangs enables the creation of two types of immiscible DNA droplets. Adhesion between the droplets can be tuned by the addition of "cross-linker NSs" that have two overhang sequences of each type. We find that increasing the amount of the cross-linker NSs decreases the droplet/droplet surface tension until a microemulsion transition occurs. Controlled droplet adhesion can also be achieved, without cross-linkers, using overhangs that can weakly hybridize. Finally, we show that solutes can be specifically targeted to the DNA phases by labeling them with appropriate sticky-ends. Overall, our findings demonstrate the ability to create a multiphase LLPS system, and to control its mesoscale configuration, via sequence design of the component molecules.

Pattern Regulation of DNA Hydrogels Formed by Lateral Phase Separation of DNA Nanostructures on Water-in-Oil Droplet Interfaces

“…Since the X-linker linked the Y-and orth Y-motifs, it can be regarded as a surfactant between the two motifs. This surfactant-like role has been confirmed in previous studies in bulk solution (29,30). Jeon et al, discussed that DNA liquid composed of DNA nanostars can form microemulsions in another DNA liquid using cross-linker nanostars (29).…”

“…This surfactant-like role has been confirmed in previous studies in bulk solution (29,30). Jeon et al, discussed that DNA liquid composed of DNA nanostars can form microemulsions in another DNA liquid using cross-linker nanostars (29). In addition, they discussed that the cross-linkers formed micelles in DNA liquids where the cross-linker nanostars were distributed in the bulk DNA liquid.…”

“…Sequence-specific DNA interactions can be used to control the phase separation of DNA nanostructures at the molecular level (28)(29)(30)(31)(32)(33). Recent studies have demonstrated that sequencedesigned DNA nanostructures exhibit temperature-dependent phase separation and self-assembly into liquid-like droplets (28)(29)(30)(31)(32). The DNA nanostructures formed droplets under a specific temperature and the formed droplets became hydrogels when the temperature was lowered (30).…”

“…The DNA nanostructures formed droplets under a specific temperature and the formed droplets became hydrogels when the temperature was lowered (30). Furthermore, two distinct liquid DNA phases were formed on the two types of DNA nanostructures with orthogonal sequence pairs (29,30), allowing the formation of two immiscible DNA liquid droplets and hydrogels. Designing a DNA-based phase separation system is a feasible approach to the design and control of lateral phase separation of compartmental capsules.…”

DNA nanotechnology provides an avenue for the construction of programmable dynamic molecular systems

“…In recent years, LLPS has attracted considerable attention in the fields of biology and biophysics, since the formation of biological condensates, including droplet-and gel-like structures, inside living cells via LLPS has been implicated in various biological processes [4,5,49,50]; however, the detailed mechanisms of biomolecular LLPS are not yet fully understood. LLPS studies based on DNA nanotechnology [48,51,52] provide a tool to simulate biological phase separation, thereby allowing investigation of the mechanism of macromolecular droplet formation, such as the effects of multivalent interaction [53] or sequence-dependency of nucleic acids on phases separation [54].…”