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

Abstract

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.

Multifaceted Cargo Recruitment and Release from Artificial Membraneless Organelles

“…The minimalist cationic AMLO harbors sites with multiple molecular interactions including electrostatic attraction, π–π and cation–π interactions, [ 15 ] having the potential to be carriers of a wide spectrum of cargoes (illustrated as Figure S1, Supporting Information). The AMLOs formed in intracellular physiological‐mimicking buffer (IPM buffer, see Supporting Information) and fetal bovine serum (FBS) cell culture media, which can stabilize mostly up to 24 h (Figure S22, Supporting Information), with acceptable cell viability under sub‐µ m amount of incorporation (Figure S21, Supporting Information).…”

“…The fluorescence intensity regenerated 60.0%, 61.8%, and 47.1% of the original value after 1, 2, and 6 cycles of heating-cooling, respectively, presumably owing to the incomplete reversibility of LLPS of IPH*. [15] macromolecules, proteins, DNAs, and RNAs. Triggered release by temperature, pH, and salt was demonstrated owing to the dynamic nature of the AMLO.…”

“…The minimalist cationic AMLO harbors sites with multiple molecular interactions including electrostatic attraction, π-π and cation-π interactions, [15] having the potential to be carriers of a wide spectrum of cargoes (illustrated as Figure S1, Supporting Information). The AMLOs formed in intracellular…”

“…As natural MLOs are hubs of myriad biological molecules engaged with complex cellular metabolism, the compositional control is of vital importance. [12] We thus examined whether environmentally responsive AMLO [15] can achieve triggered recruitment or release of biomolecules, by studying two model biomolecules: BSA and dsDNAs. Three stimuli-temperature, pH, and ionic strength (salt concentration)-were investigated, owing to their relevance to physiology [28][29][30][31] and significant contributions to the phase behavior.…”

“…IPH*, which is designed to mimic FUS protein, phase separated to form artificial MLOs, which could preferentially enrich green fluorescence protein and model RNAs in the interior. [15,16] The liquid nature of artificial MLO (AMLO) is ideal for the hosting and preservation of biological molecules, affording the possibility of sensing, as well as fast, adaptive, and reversible responses to various stimuli (e.g., pH and heat) in solution. [17] As such, the AMLO with liquidity can be advantageous over solid particles for applications such as controlled release and biosensing.…”