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Abstract RNA-driven phase separation is emerging as a promising approach for engineering biomolecular condensates with diverse functionalities. Condensates form thanks to weak yet specific RNA–RNA interactions established by design via complementary sequence domains. Here, we demonstrate how RNA condensates formed by star-shaped RNA motifs, or nanostars, can be dynamically controlled when the motifs include additional linear or branch-loop domains that facilitate access of regulatory RNA molecules to the nanostar interaction domains. We show that condensates dissolve in the presence of RNA “invaders” that occlude selected nanostar bonds and reduce the valency of the nanostars, preventing phase separation. We further demonstrate that the introduction of “anti-invader” strands, complementary to the invaders, makes it possible to restore condensate formation. An important aspect of our experiments is that we demonstrate these behaviors in one-pot reactions, where RNA nanostars, invaders, and anti-invaders are simultaneously transcribed in vitro using short DNA templates. Our results lay the groundwork for engineering RNA-based assemblies with tunable, reversible condensation, providing a promising toolkit for synthetic biology applications requiring responsive, self-organizing biomolecular materials.
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Internal Phase Separation in Synthetic DNA Condensates
Abstract Biomolecular condensates regulate cellular biochemistry by organizing enzymes, substrates and metabolites, and often acquire partially de‐mixed states whereby distinct internal domains play functional roles. Despite their physiological relevance, questions remain about the principles underpinning the emergence of multi‐phase condensates. Here, a model system of synthetic DNA nanostructures able to form monophasic or biphasic condensates is presented. Key condensate features, including the degree of interphase mixing and the relative size and spatial arrangement of domains, can be controlled by altering nanostructure stoichiometries. The modular nature of the system facilitates an intuitive understanding of phase behavior, and enables mapping of the experimental phenomenology onto a predictive Flory‐Huggins model. The experimental and theoretical framework introduced is expected to help address open questions on multiphase condensation in biology and aid the design of functional biomolecular condensates in vitro, in synthetic cells, and in living cells.
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- Award ID(s):
- 2134772
- PAR ID:
- 10640167
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Advanced Science
- Volume:
- 12
- Issue:
- 41
- ISSN:
- 2198-3844
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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