Emergence of Anisotropic Subcompartments via Co-Assembly of Hierarchically Ordered G-Quadruplexes and Dynamic Polylysine in Droplet-Based Compartments

Abstract

Liquid–liquid phase separation (LLPS) plays a key role in cellular organization, enabling the formation of dynamic compartments that provide spatial and temporal control over biochemical processes. Although LLPS systems are typically fluidic, recent studies have revealed that specific molecular constituents can induce internal structure. Here, we show that droplet formation between guanine quadruplexes (G4s) and polylysine peptides triggers G4-driven internal structural ordering. Using birefringence-retardation imaging, confocal fluorescence imaging, as well as X-ray diffraction and scattering techniques, we demonstrate that anisotropic subcompartments are gradually developed at specific points of their droplet–solution interfaces. The G4s possess intrinsic molecular rigidity due to their stacked G-quartet structures, and their interaction with the flexible polylysine peptides enables the formation of a hexagonal columnar phase (a = 3.64 nm; c = 0.86 nm; nine units per turn). This highly ordered subcompartment is enriched in low-fluidity G4s (τ > 103 s), while the peptides remain dynamically distributed throughout the entire compartment (τ ≈ 6.6 s). This emergence of multiphase organization within a single compartment is reminiscent of the hierarchical structure of membrane-less organelles. Moreover, the coexistence of aligned low fluidity and diffusing components within the same space evokes raft-like domains. This supramolecular platform provides insights into the cooperative roles of structural order and molecular mobility in phase-separated systems, offering a foundation for the bottom-up design of synthetic condensates inspired by biomolecular organization.

Affiliated Institutions

• Shanghai Institute for Science of Science CN
• Institute of Science Tokyo
• National Institute of Advanced Industrial Science and Technology JP
• Tohoku University JP

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