Abstract
Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.
Highlights
Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease
We find that polyK cannot, form droplets with uridine-5′driphosphate disodium salt (UDP) or uridine-5′-monophosphate (UMP) under the conditions tested (Supplementary Fig. 1)
How do sequence-level changes influence condensate material properties; how do material properties in turn influence condensate dynamics, multiphase coexistence, and function; and can bottom–up sequence design rules be generated to engineer condensates with specific material properties that can be leveraged for controlling condensate dynamics
Summary
Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. Recent fluorescence recovery after photobleaching (FRAP) studies indicate that proline–arginine dipeptide repeats implicated in ALS give rise to condensates that have less internal mobility than comparable proline–lysine dipeptide repeats[40], with similar observations of reduced fluidity made for model arginine–glycine vs lysine–glycine peptide sequences[41] and lysine and argininerich peptides[42] These recent works have shown that substituting poly-RNA bases (purine vs pyrimidine) had distinct consequences on the apparent fluidity of respective arginine- vs lysine-rich peptides. Direct rheological measurements comparing arginine and lysine homopolymer condensates would provide fundamental insight into how these residues contribute to network properties, such as viscosity Material properties such as viscosity and surface tension dictate many essential characteristics of condensates, including internal diffusion rates, molecular sequestration, and the hierarchical organization of coexisting phases[43,44]. The sequence-driven rules underlying the multiphase droplet formation of charged biopolymers are just beginning to be unraveled
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