Condensates formed via liquid-liquid phase separation (LLPS) provide a chemically versatile environment for catalysis through dynamic molecular interactions. We present designed biomolecular condensates, formed by LLPS of minimalistic histidine-containing peptides, catalyzing ester hydrolysis with two distinct mechanisms. Zn2+-dependent condensates activate a coordinating water molecule at the active site, formed by Zn2+-histidine coordination, enabling nucleophilic attack. We show that dense-phase basicity, internal mobility, and Zn2+ accumulation within the condensates collectively govern their catalytic activity. In the absence of Zn2+, catalysis is driven by intermolecular low-barrier hydrogen bonds between histidine residues, facilitating nucleophile formation. Combined computational and experimental evidence reveals the molecular basis of these catalytic pathways, demonstrating the functionality of biomolecular condensates in catalysis and nanotechnology. These findings establish a foundation for exploring mechanisms of metal-free emergent catalysis within complex liquid assemblies, expanding the potential of LLPS-based systems in green chemistry and advanced materials.

Nano- and microencapsulation that combines high loading capacity with stimulus-responsive release remains a challenge for therapeutic delivery. Designed peptide condensates formed by liquid-liquid phase separation offer a versatile and biocompatible platform to address this need. Here, we systematically link backbone flexibility and ion identity to phase behavior, material properties, payload encapsulation, and protease-triggered condensate disassembly using minimalistic cationic-aromatic peptides that differ in their glycine content. Moreover, we systematically studied how monovalent vs divalent anions affect phase behavior and payload encapsulation. Our results show that glycine-poor sequence forms the most highly packed condensates and that the kosmotrope divalent sulfate ions markedly increase dense-phase peptide concentration and droplet size. Glycine content, which regulates charge density and backbone flexibility, directly affects condensate dynamics, showing faster diffusion with an increasing number of glycine residues. High-performance liquid chromatography partitioning analysis of five FDA-approved small molecules demonstrates compound-specific and salt-mediated recruitment, where both the hydrophobicity/polarity and the charge state of the compounds affect their encapsulation in the dense phase. Moreover, using trypsin as a proteolytic trigger, we show how the glycine content affects condensate disassembly. Overall, these results facilitate practical design rules that show how to tune charge and aromatic density, backbone flexibility, and ion valency to regulate dense-phase packing, payload encapsulation, and release. These insights advance the rational engineering of peptide condensates for targeted sequestration and controlled therapeutic release.

Biomolecular condensates, formed through liquid-liquid phase separation, serve as dynamic platforms for biochemical regulation. Inspired by these natural systems, we develop designer peptide-based condensates to modulate chemical transformations, focusing on the Cu(I)-catalyzed azide-alkyne cycloaddition click reaction between hydrophobic reactants as a model system. By incorporating a varying number of isoleucine residues into peptide sequences, we tune the hydrophobicity of the condensates. This variation allows us to tune condensate properties, including reactant recruitment, internal mobility, and catalytic performance. We show that peptide hydrophobicity dictates selective partitioning of the hydrophobic azide reactant into the dense phase, while increased hydrophobicity reduces internal diffusion. Higher molecular mobility within the condensates correlates with increased reaction rates and product formation, leading to enhanced spatially localized reactivity within the condensates. Together, our findings establish a mechanistic framework linking the peptide sequence, condensate dynamics, and compartmentalized catalysis. This work provides a foundation for using designer condensates as programmable microreactors for sustainable chemistry and biomedical applications.

Membraneless compartments formed by liquid-liquid phase separation (LLPS) regulate biochemical reactions and play a key role in both physiological and pathological processes, including viral replication. In retroviral systems, the extent of genome folding is critical for the efficient packaging of new viral particles, a process mediated by the nucleocapsid (NC) protein that chaperones RNA folding and assembly. Here, we sought to elucidate how nucleic-acid folding and structural folding influence LLPS and whether an HIV NC-derived peptide (HNP) can modulate this process through chaperone-like activity. To this end, we designed a programmable single-stranded DNA (ssDNA) library spanning varying degrees of folding and palindromic architectures, enabling systematic investigation of how nanoscale structural order governs coacervation. Using circular dichroism, FRET, SAXS, and coarse-grained simulations, we correlate DNA conformations with phase behavior and emergent condensate material properties. We find that interactions with HNP promote DNA folding and that increasing DNA order suppresses LLPS, whereas structural disorder and palindromic linkers that induce DNA dimerization enhance phase separation by facilitating multivalent interactions and in turn increasing condensate viscosity. Together, these findings identify two programmable determinants, local structural order and palindromic dimerization, that govern DNA/peptide condensate behavior, offering mechanistic insight into viral genome organization and guiding principles for tuning the physicochemical and material properties of synthetic condensates.

Synthetic biomolecular condensates, formed through liquid-liquid phase separation (LLPS), are increasingly studied us-ing RNA and DNA polymers as fundamental building blocks. While RNA/peptide LLPS is well characterized, the phase behavior of DNA and its interactions with peptides remain largely unexplored. Inspired by the viral machinery that al-lows the packaging of large nucleic acid polymers into nanometer-sized compartments¬ facilitated by specific nucleic acid-amino acid interactions¬ we investigated how minimalistic peptides modulate the structural order of single-stranded DNA (ssDNA) to regulate condensate formation. We engineered two ssDNA variants, an ordered ssDNA with tRNA-like struc-ture and another largely disordered and combined experimental and computational approaches to analyze their phase behavior. Our results show that ordered DNA restricts LLPS, whereas disordered DNA enhances condensate density and slows molecular diffusivity. These findings establish a direct link between DNA order/disorder and mesoscale phase separation, expanding our understanding of nucleic acid-driven LLPS and offering new strategies for designing pro-grammable DNA-peptide condensates with tunable properties.

Designed biomolecular condensates are emerging condensed-phase assemblies, initially conceived to mimic cellular biomolecular condensates for use in biology-inspired applications such as delivery and storage of biomolecules. In recent years, rational design approaches informed by supramolecular chemistry and biomolecular nanotechnology, including the use of peptide and DNA nanotechnology for building-block minimalization and site-specific interactions, have evolved rapidly, going beyond the molecular basis of cellular condensates in terms of both composition and functionality. Thus, synthetic condensates are designed from diverse molecular building blocks, including single- or multicomponent polypeptides, peptides, RNA, DNA or biopolymers; moreover, their applications are continuously evolving to encompass new nanotechnology-relevant functions including biosensing and bioadhesion, where condensates offer advantages such as responsiveness, programmability and molecular compartmentalization. In this Review, we show the main concepts behind the molecular design of synthetic condensates, from biological mimicry to purely synthetic approaches. We discuss the mechanisms that allow control and regulation of condensate properties and the remaining challenges in analysing these properties. Finally, we discuss the applications of synthetic condensates thus far, the potential in leveraging condensates as platforms for nanotechnological applications, and the remaining hurdles towards realizing this promise. We also provide an overview of the patent landscape, highlighting trends in commercial development across areas such as delivery systems, microreactors and sensing technologies.

Condensates formed via liquid-liquid phase separation provide a chemically versatile environment for catalysis through dynamic molecular interactions. We present designed biomolecular condensates, formed by LLPS of minimalistic histidine-containing peptides, catalyzing ester hydrolysis with two distinct mechanisms. Zn2+-dependent condensates activate a coordinating water molecule at the active site, formed by Zn2+-histidine coordination, enabling nucleophilic attack. In the absence of Zn2+, catalysis is driven by intermolecular low-barrier hydrogen bonds between histidine residues, facilitating nucleophile formation. Combined computational and experimental evidence reveals the molecular basis of these catalytic pathways, demonstrating the functionality of biomolecular condensates in catalysis and nanotechnology. These findings establish a foundation for exploring new mechanisms of metal-free emergent catalysis within complex liquid assemblies, expanding the potential of LLPS-based systems in green chemistry and advanced materials.

In the presence of complementary short oligonucleotide strands within synthetic DNA condensates, a striking mode of molecular transport is observed, revealing a sharp, wave-like diffusion front driven by phase-swelling effects and transitions in the material state of the condensates.

Compartmentalization, a key aspect of biochemical regulation, naturally occurs in cellular organelles, including biomolecular condensates formed through liquid-liquid phase separation (LLPS). Inspired by biological compartments, synthetic coacervates have emerged as versatile microreactors, which can provide customed environments for enzymatic reactions. In this review, we explore recent advances in coacervate-based microreactors, while emphasizing the mechanisms by which coacervates accelerate enzymatic reactions, namely by enhancing substrate and enzyme concentrations, stabilizing intermediates, and providing molecular crowding. We discuss diverse coacervate systems, including those based on synthetic polymers, peptides, and nucleic acids, and describe the selection of enzymatic model systems, as well as strategies for enzyme recruitment and their impact on reaction kinetics. Furthermore, we discuss the challenges in monitoring reactions within coacervates and review the currently available techniques including fluorescence techniques, chromatography, and NMR spectroscopy. Altogether, this review offers a comprehensive perspective on recent progress and challenges in the design of coacervate microreactors, and addresses their potential in biocatalysis, synthetic biology, and nanotechnology.

Membraneless organelles are cellular biomolecular condensates that are formed by liquid-liquid phase separation (LLPS) of proteins and nucleic acids. LLPS is driven by multiple weak attractive forces, including intermolecular interactions mediated by aromatic amino acids. Considering the contribution of pi-electron bearing side chains to protein-RNA LLPS, systematically study sought to how the composition of aromatic amino acids affects the formation of heterotypic condensates and their physical properties. For this, a library of minimalistic peptide building blocks is designed containing varying number and compositions of aromatic amino acids. It is shown that the number of aromatics in the peptide sequence affect LLPS propensity, material properties and (bio)chemical stability of peptide/RNA heterotypic condensates. The findings shed light on the contribution of aromatics' composition to the formation of heterotypic condensates. These insights can be applied for regulation of condensate material properties and improvement of their (bio)chemical stability, for various biomedical and biotechnological applications. Using designer peptides, it is shown that how aromatic amino acids influence the phase behavior and properties of heterotypic peptide/RNA biomolecular condensates, including peptide diffusion and condensate chemical and biochemical stability. These findings can aid in constructing more durable condensates for diverse biotechnological uses. image

Biomolecular condensates are condensed intracellular phases that are formed by liquid-liquid phase separation (LLPS) of proteins, either in the absence or presence of nucleic acids. These condensed phases regulate various biochemical reactions by recruitment of enzymes and substrates. Developments in the field of LLPS facilitated new insights on the regulation of compartmentalized enzymatic reactions. Yet, the influence of condensate chemical composition on enzymatic reactions is still poorly understood. Here, by using peptides as minimalistic condensate building blocks and β-galactosidase as a simple enzymatic model we show that the reaction is restricted in homotypic peptide condensates, while product formation is enhanced in peptide-RNA condensates. Our findings also show that condensate composition affects the recruitment of substrate, the spatial distribution, and the kinetics of the reaction. Thus, these findings can be further employed for the development of microreactors for biotechnological applications.

Viral assembly, similar to other self-organizing protein systems, relies upon early building blocks, which associate into the late supramolecular structures. An initial and crucial event during HIV-1 core assembly is the dimerization of the capsid protein C-terminal domain, which stabilizes the viral capsid lattice. Thus, monitoring and manipulating this stage is desirable both from mechanistic as well as clinical perspectives. Here, we developed a fluorescent-based method for the detection and visualization of these early capsid interactions. We detected strong dimeric interactions, which were influenced by mutations in the capsid protein. We utilized this assay for potential assembly inhibitors screening, which resulted in the identification of a leading compound that hinders the assembly of capsid protein in vitro. Moreover, a derivative of the compound impaired virus production and infectivity in cell cultures. These findings demonstrate that the described assay efficiently detects the very first association events in HIV-1 capsid formation and emphasize the significance of targeting early intermolecular interactions.

Biomolecular condensates, formed through liquid-liquid phase separation (LLPS), serve as enzymatic reaction centers in cells by increasing local concentrations of enzymes and substrates, thereby facilitating reaction kinetics and regulatory mechanisms. Inspired by these natural systems, synthetic condensates are being developed for diverse applications, including payload delivery, sensing, and as microreactors where enzymatic reaction kinetics can be modulated by factors like pH, viscosity, and enzyme-substrate co-localization. Here, we investigate how the physicochemical properties of enzymes and substrates influence condensate formation and function as microreactors. Focusing on cellulase and alkaline phosphatase, which differ in molecular weight and isoelectric point, we employed a minimalistic complex coacervation system of oppositely charged LLPS-promoting peptides. Our findings show how electrostatic forces within condensates influence their role as microreactors. Specifically, the ability of condensates to encapsulate or exclude phosphatase, cellulase, and their substrates, which is pivotal for the regulation of reaction kinetics, is determined by the enzyme surface charge, substrate charge, and condensate charge stoichiometry. These results highlight the potential of utilizing electrostatic forces within condensates to modulate enzymatic reactions, providing critical insights for developing synthetic condensates as microreactors in biotechnology and materials science.

The dynamic nature of cellular microenvironments, regulated by the viscoelasticity and enzymatic cleavage of the extracellular matrix, remains challenging to emulate in engineered synthetic biomaterials. To address this, a novel platform of cell-instructive hydrogels is introduced, composed of two concurrently forming interpenetrating polymer networks (IPNs). These IPNs consist of the same basic building blocks - four-armed poly(ethylene glycol) and the sulfated glycosaminoglycan (sGAG) heparin - are cross-linked through either chemical or physical interactions, allowing for precise and selective tuning of the hydrogel's stiffness, viscoelasticity, and proteolytic cleavability. The studies of the individual and combined effects of these parameters on stem cell behavior revealed that human mesenchymal stem cells exhibited increased spreading and Yes-associated protein transcriptional activity in more viscoelastic and cleavable sGAG-IPN hydrogels. Furthermore, human induced pluripotent stem cell (iPSC) cysts displayed enhanced lumen formation, growth, and pluripotency maintenance when cultured in sGAG-IPN hydrogels with higher viscoelasticity. Inhibition studies emphasized the pivotal roles of actin dynamics and matrix metalloproteinase activity in iPSC cyst morphology, which varied with the viscoelastic properties of the hydrogels. Thus, the introduced sGAG-IPN hydrogel platform offers a powerful methodology for exogenous stem cell fate control.

Inspired by the role of intracellular liquid-liquid phase separation (LLPS) in formation of membraneless organelles, there is great interest in developing dynamic compartments formed by LLPS of intrinsically disordered proteins (IDPs) or short peptides. However, the molecular mechanisms underlying the formation of biomolecular condensates have not been fully elucidated, rendering on-demand design of synthetic condensates with tailored physico-chemical functionalities a significant challenge. To address this need, here we design a library of LLPS-promoting peptide building blocks composed of various assembly domains. We show that the LLPS propensity, dynamics, and encapsulation efficiency of compartments can be tuned by changes to the peptide composition. Specifically, with the aid of Raman and NMR spectroscopy, we show that interactions between arginine and aromatic amino acids underlie droplet formation, and that both intra- and intermolecular interactions dictate droplet dynamics. The resulting sequence-structure-function correlation could support the future development of compartments for a variety of applications.

Most biocatalytic processes in eukaryotic cells are regulated by subcellular microenvironments such as membrane bound or membraneless organelles. These natural compartmentalization systems have inspired the design of synthetic compartments composed of a variety of building blocks. Recently, the emerging field of liquid-liquid phase separation has facilitated the design of biomolecular condensates composed of proteins and nucleic acids, with controllable properties including polarity, diffusivity, surface tension, and encapsulation efficiency. However, utilizing phase- separated condensates as optical sensors has not yet been attempted. Here, we were inspired by the biosynthesis of melanin pigments, a key biocatalytic process that is regulated by compartmentalization in organelles, to design minimalistic biomolecular condensates with emergent optical properties. Melanins are ubiquitous pigment materials with a range of functionalities including photoprotection, coloration, and free radical scavenging activity. Their biosynthesis in the confined melanosomes involves oxidation- polymerization of tyrosine (Tyr), catalyzed by the enzyme tyrosinase. We have now developed condensates that are formed by an interaction between a Tyr- containing peptide and RNA and can serve as both microreactors and substrates for tyrosinase. Importantly, partitioning of Tyr into the condensates and subsequent oxidation- polymerization gives rise to unique optical properties including far red fluorescence. We now demonstrate that individual condensates can serve as sensors to detect tyrosinase activity, with a limit of detection similar to that of synthetic fluorescent probes. This approach opens opportunities to utilize designer biomolecular condensates as diagnostic tools for various disorders involving abnormal enzymatic activity.

Here, we utilized designed condensates formed by liquid-liquid phase separation (LLPS) of cationic and aromatic peptide to sequester tyrosine-based carbon dots (C-dots). The C-dots fluorescence is quenched and retrieved upon partitioning and release from condensates, allowing a spatial regulation of C-dots fluorescence which can be utilized for biosensing applications.

In the past two decades, peptides and proteins have been studied extensively in many fruitful directions. Peptides and proteins can serve as building blocks for functional materials. They can self-assemble into various objects such as tubes, spheres, fibrils, and hydrogels and can be chemically and biologically modified to serve a specific function. Their role at the origin of life as self-replicating molecules or catalysts has been greatly explored. In addition, since protein-protein and peptide-protein interactions serve a key role in cancer, neurodegenerative diseases, and viral/bacterial infections, this aspect has been widely investigated to promote drug development. Gaining insights into protein-protein interactions and peptide self-assembly requires developing high-resolution research tools. This includes computational and experimental tools for structure analysis, biochemical assays, and high-resolution biophysical tools such as SAXS and XRD. Intending to cover this broad research area, we launched this special issue titled “Peptide and Protein Self-Assembly and Interactions.” The issue includes a collection of review and research articles that focus on a wide range of topics, including peptide nanomaterials and peptide-protein assemblies for (bio)medical and electronic applications, peptide structures with catalytic capacities, and multicomponent peptide-polymer, peptide-metal ions, and peptide-nucleic acid systems. Peptide-based materials are attractive for biomedical applications due to their biocompatibility and diversity. Two research papers in this special issue demonstrate two distinctive approaches that utilize peptide assemblies to fight cancer. Das and his co-workers (DOI: 10.1002/ijch.202200001) synthesized and studied the assembly of two charged cell penetrating tripeptides. They demonstrated the encapsulation of chemotherapeutic drugs by peptide assemblies and showed how they can facilitate intracellular drug delivery via endocytosis. Gosh et al. (DOI: 10.1002/ijch.202200019) crafted a peptide that promotes tubulin depolymerization and self-assembles into vesicles. These vesicles can act as a drug delivery vehicle of anti-cancer drugs. Leveraging the interface between peptide assemblies and proteins opens new exciting opportunities for medical applications. In their review paper, Marchesan and co-workers (Mañas-Torres et al, DOI: 10.1002/ijch.202200018) describe recent efforts to develop peptide assemblies as protein inhibitors, crystallization inducers, and vaccine components. This work is featured on the front cover of this issue. The interface between peptides and proteins can be harnessed as a therapeutic strategy by blocking key cellular processes through interference with the interactions between proteins and their ligand partners. Friedler and co-workers (Solomon et al., DOI: 10.1002/ijch.202200041) demonstrate this approach for inhibiting kinase activity as an anti-cancer strategy. Owing to the rich chemical repertoire of the 20-gene encoded amino acids, the organization and display of their side chain groups within ordered peptide nanostructures have been utilized for diverse functionalities, including electronic and catalytic abilities. The latter is typically achieved by a hierarchical display of reactive side chain groups that mimic enzymes‘ reactive sites. In their review article, Pina and co-workers (Carvalho et al., DOI: 10.1002/ijch.202200029) discuss recent attempts to discover catalytic function in short peptides. Incorporating non-canonical amino acids or amino acids conjugated to synthetic protecting groups to self-assembling peptides can further increase the chemical repertoire of the canonical amino acids and is widely used to enhance certain functionalities of molecular peptide materials. Gazit and co-workers (Finkelstein-Zuta et al., DOI: 10.1002/ijch.202200027) report on a piezoelectric material composed of ordered layer of nanostructures formed by non-canonical dipeptide conjugated to a protecting group, in which the molecular organization of the peptide at the nanoscale dictates the piezoelectric response. Biological materials differ from their synthetic counterparts in their complexity, as the latter are typically formed by a homogenous population of identical building blocks while the first rely on interactions between multiple building blocks, including proteins, polysaccharides and nucleic acids. Inspired by the chemical complexity and rich set of properties of these biological complexes, increasing efforts have been made toward developing multicomponent supramolecular materials. In their review article, Yosefi and Bitton (DOI: 10.1002/ijch.202200008) highlight recent work on designed hierarchical membranes formed by interactions between self-assembling peptides and biological polymers to mimic the properties of the extracellular environment. An additional class of multicomponent materials that have similar nanomechanical properties to those of the natural extracellular matrix are metallogels, hydrogels that are formed by interaction between metals and peptides or proteins. Ghrayeb and Chai (DOI: 10.1002/ijch.202200011) review key examples of peptide and protein-based metallogels, their formation mechanism, properties, and utilities. Beyond applications in nanobiotechnology, focusing on multicomponent materials and on the emergent properties of multicomponent complexes is highly relevant to the field of systems chemistry and can facilitate insights into the origin of life, especially concerning studying molecular aspects of multicomponent complexes using system-level approaches. Ashkenasy and co-workers (DOI: 10.1002/ijch.202200030) cover recent work on peptide/nucleic acid co-assembly, which until recently were utilized individually as building blocks of supramolecular materials. The review highlights work on virus-inspired peptide/nucleic acid structures, dynamic assemblies formed by peptide/nucleic acid chimeras, functional peptide/nucleic acid materials, self-replicators, and nucleic acid-(depsi)peptide networks, in the context of systems chemistry. In summary, this themed issue attempts to cover the widely ranged and rapidly evolving area of peptide and protein self-assembly. Obviously, we have not covered a variety of topics in this field but rather focused on peptide assemblies and protein-peptide interactions for medical applications, peptide nanomaterials with electronic and catalytic functions, and multicomponent peptide materials and complexes. We are grateful to all contributors to this issue, who brought their unique angles and covered various aspects of the field.

Viral factories are intracellular microcompartments formed by mammalian viruses in their host cells, and contain necessary machinery for viral genome replication, capsid assembly, and maturation, thus serving as “factories” for formation of new viral particles. Recent evidence suggests that these compartments are formed by liquid–liquid phase separation (LLPS) of viral proteins and nucleic acids and present dynamic properties. In this work, inspired by the remarkable functionalities of viral factories, dynamic compartments that are formed by complexation between a minimalistic, disordered peptide and RNA are designed. By systematic studies using sequence variants it is shown that the material properties of the compartments can be modulated by changes to the peptide sequence, at the single amino acid level. Moreover, by taking this approach to the next step, liquid compartments with light‐induced tunable dynamics are developed. The results demonstrate that the material properties of liquid droplets can be temporally regulated by increasing peptide polarity and charge, and that these changes can be further utilized for controlled partitioning and release of payloads from the compartments.