Design and Characterization of DNA-Driven Condensates: Regulating Topology, Mechanical Properties, and Immunorecognition

Cells maintain spatiotemporal control over biochemical processes through the formation and dissolution of biomolecular condensates, dynamic membraneless organelles formed via liquid-liquid phase separation. Composed primarily of proteins and nucleic acids, these condensates regulate key cellular functions, and their properties are influenced by the concentration and type of molecules involved. The structural versatility challenges the <i>de novo</i> design and assembly of condensates with predefined properties. Through feedback between computational and experimental approaches, we introduce a modular system for assembling condensates using nucleic acid nanotechnology. By utilizing programmable oligonucleotides and orthogonal synthesis methods, we control the structural parameters, responsive behavior, and immunorecognition of the products. Dissipative particle dynamics simulations predict some conditions to produce larger, well-defined condensates with compact, globular cores, while others result in smaller, more diffuse analogs. Fluorescence microscopy confirms these findings and microrheology demonstrates the viscoelastic adaptability of tested condensates. Nucleases trigger disruption of structures, and ethidium bromide intercalation protects condensates from digestion. Immunostimulatory assays suggest condensate-specific activation of the IRF pathway via cGAS-STING signaling. This study provides a framework for developing biomolecular condensates with customizable properties and immunorecognition for various biological applications.