Construction of multiphasic membraneless organelles towards spontaneous spatial segregation and directional flow of biochemical reactions.

Many intracellular membraneless organelles (MLOs) appear to adapt a hierarchical multicompartment organization for efficient coordination of highly complex reaction networks. Recapitulating such an internal architecture in biomimetic platforms is, therefore, an important step to facilitate the functional understanding of MLOs and to enable the design of advanced microreactors. Herein, we present a modular bottom-up approach for building synthetic multiphasic condensates using a set of engineered multivalent polymer-oligopeptide hybrids. These hybrid constructs exhibit dynamic phase separation behaviour generating membraneless droplets with a subdivided interior featuring distinct chemical and physical properties, whereby a range of functional biomolecules can be spontaneously enriched and spatially segregated. The platform also attains separated confinement of transcription and translation reactions in proximal compartments, while allowing inter-compartment communication via a directional flow of reactants. With advanced structural and functional features attained, this system can be of great value as a MLO model and as a cell-free system for multiplex chemical biosynthesis.

Nucleic Acids Modulate Liquidity and Dynamics of Artificial Membraneless Organelles.

Liquid-liquid phase separation (LLPS) emerges as a fundamental underlying mechanism for the biological organization, especially the formation of membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Nucleic acids are compositional biomacromolecules of MLOs with wide implications in normal cell functions as well as in pathophysiology caused by aberrant phase behavior. Exploiting a minimalist artificial membraneless organelles (AMLO) from LLPS of IDP-mimicking polymer-oligopeptide hybrid (IPH), we investigated the effect of nucleic acids with different lengths and sequence variations on AMLO. The behavior of this AMLO in the presence of DNAs and RNAs resembled natural MLOs in multiple aspects, namely, modulated propensity of formation, morphology, liquidity, and dynamics. Both DNA and RNA could enhance the LLPS of AMLO, while compared with RNA, DNA had a higher tendency to solidify and diminish dynamics thereof. These findings suggest its potential as a concise model system for the understanding of the interaction between nucleic acids and natural MLOs and for studying the molecular mechanism of diseases involving MLOs.

Multifaceted Cargo Recruitment and Release from Artificial Membraneless Organelles.

Liquid-liquid phase separation (LLPS) drives membraneless organelles (MLOs) formation for organizing biomolecules. Artificial MLOs (AMLOs) have been constructed mostly via the LLPS of engineered proteins capable of regulating limited types of biomolecules. Here, leveraging a minimalist AMLO, driven by LLPS of polymer-oligopeptide hybrids, enrichment, recruitment, and release of multifaceted cargoes are quantitatively shown, including small fluorescent molecules, fluorophore-containing macromolecules, proteins, DNAs, and RNAs. Cargoes show up to 105 -fold enrichment, whilst recruitment and release are triggered by variations of temperature, pH, and/or ionic strength. Also, the first efficacious, rapid, and reversible control of aggregation-induced emission with over 30 folds of modulation of overall fluorescence intensity is achieved, by intensifying the aggregation of luminogens in AMLO. The AMLO is a simple yet versatile platform for potential drug delivery and biosensor applications.

Minimalist Design of an Intrinsically Disordered Protein-Mimicking Scaffold for an Artificial Membraneless Organelle.

Liquid-liquid phase separation (LLPS) is an emerging and universal mechanism for intracellular organization, particularly, by forming membraneless organelles (MLOs) hosting intrinsically disordered proteins (IDPs) as scaffolds. Genetic engineering is generally applied to reconstruct IDPs harboring over 100 amino acid residues. Here, we report the first design of synthetic hybrids consisting of short oligopeptides of fewer than 10 residues as "stickers" and dextran as a "spacer" to recapitulate the characteristics of IDPs, as exemplified by the multivalent FUS protein. Hybrids undergo LLPS into micron-sized liquid droplets resembling LLPS in vitro and in living cells. Moreover, the droplets formed are capable of recruiting proteins and RNAs and providing a favorable environment for a biochemical reaction with highly enriched components, thereby mimicking the function of natural MLOs. This simple yet versatile model system can help elucidate the molecular interactions implicated in MLOs and pave ways to a new type of biomimetic materials.