Macromolecular crowding helps control synthetic DNA droplet formation

Macromolecular crowding can serve as a design tool for controlling the formation and organization of synthetic DNA droplets, a study from Science Tokyo reveals. Researchers investigated this novel strategy using DNA nanostars and polymeric crowding agents to mimic the crowded cellular environment. They found that crowding promoted droplet formation and enabled control over droplet size, interactions, and organization. The findings could support the development of programmable artificial cell systems.

Biomolecules, such as proteins and nucleic acids, can gather into concentrated regions called biomolecular condensates. These condensates regulate various cellular processes. However, the cell interior is also crowded with other macromolecules that can influence the properties of biomolecular condensates. Therefore, researchers are interested in understanding how this crowded environment, exhibiting a phenomenon known as macromolecular crowding, affects the formation, stability, and organization of these droplets.

Scientists from Institute of Science Tokyo (Science Tokyo), Japan, have investigated this aspect using synthetic Y-shaped branched DNA nanostructures called DNA nanostars, which self-assemble into liquid-like DNA droplets. They created a molecularly crowded environment that mimics the interior of a living cell using polymeric crowding agents and examined how this environment affected the formation, stability, and organization of these DNA droplets.

The study was led by Professor Masahiro Takinoue, together with graduate student Naoki C. Yoshida from Science Tokyo (at the time of the study), Dr. Mayu Shono at The University of Tokyo, Japan, and Professor Kenichi Yoshikawa of Doshisha University, Japan. This paper was made available online on July 27, 2026, and was published in Volume 6, Issue 8 of the journal JACS Au on August 24, 2026.

We provide a new design strategy for constructing sophisticated artificial cell architectures and molecular networks based on programmable self-organization."

Professor Masahiro Takinoue, Science Tokyo

Nanostars connect through short complementary DNA sequences called sticky ends. As the nanostars interact with one another, they can undergo liquid–liquid phase separation, producing a concentrated, liquid-like DNA phase that appears as droplets. Without crowding agents, the researchers observed droplet formation below 63 °C as they cooled the solution from 85 °C. Adding crowding agents promoted droplet formation at higher temperatures. When the researchers added polyethylene glycol (PEG), dextran, or Ficoll at a concentration of 10%, the droplets formed at higher temperatures of 76, 65, and 67 °C, respectively. This showed that molecular crowding helped the droplets form more easily and remain stable at higher temperatures.

Among the crowding agents, PEG with a high molecular weight of 7.5 kDa had the most prominent effect, so the researchers selected it for further experiments to investigate how crowding affects DNA condensate formation. PEG promotes droplet formation but cannot work alone. When added to nanostars lacking sticky ends, PEG produced no condensates, showing that it requires sticky-end interactions to promote nanostar assembly.

Using PEG, the researchers demonstrated that crowding can be used to organize different DNA droplets into specific structures. For this, they created two types of nanostars whose sticky ends could not interact with or bind to each other.

Under normal conditions, the different nanostar types formed separate droplets that did not mix or fuse with each other. However, when the researchers added PEG, the crowding effect brought the droplets into contact without causing them to fuse. By controlling the concentration of PEG, the ratio of different nanostars, and the salt concentration, the researchers could control how the droplets interacted and were organized, producing patterns ranging from alternating, network-like structures to more complex structures containing upto three distinct types of DNA nanostars.

"These findings reveal that macromolecular crowding is not merely a feature of the cellular environment but also serves as a powerful design parameter for engineering artificial DNA condensates," says Takinoue.

By harnessing macromolecular crowding, researchers could develop synthetic biomolecular systems with controlled structures and functions. "The resulting design principles are expected to contribute to the development of artificial cell tissues, molecular communication networks, molecular computing systems, and programmable micromachines, where function emerges through controlled self-organization of biomolecular components," says Takinoue.