Designer Peptides for Single-Based Functional Coacervates with Enhanced Catalytic Activity

Coacervates comprising peptides that undergo liquid-liquid phase separation (LLPS). These peptide coacervates can be tailored for applications in drug delivery, biosensing, bioelectronics, and in a range of other biotechnological purposes.

BACKGROUND

Liquid-liquid phase separation (LLPS) in living cells provides innovative pathways for synthetic compartmentalized catalytic systems. While LLPS has been explored for enhancing enzyme catalysis, its potential application to catalytic peptides remains unexplored.

Therefore, there is a need to improve the catalytic efficiency of peptides. Catalytic peptides are highly flexible molecules, which, while advantageous for adaptability, can suffer from reduced catalytic efficiency due to their conformational mobility, especially in aqueous solutions. This flexibility presents challenges in achieving consistent and efficient catalytic activity, as the lack of structure often hinders proper substrate alignment and lowers reaction rates. Thus, the technical challenge is to restrict the movement of peptides to effectively stabilize their conformations and form structured peptide domains, which will increase local reaction rates and improve overall catalytic efficiency.

This technology enables a single peptide to induce compartmentalization, regulate, and mediate specific functions. It provides proof-of-concept for establishing low-complexity, single peptide-based compartments with diverse potential applications in fields such as drug delivery, sensing, and bioelectronics.

The innovative approach of using designer peptides for creating functional, compartmentalized structures offers a versatile platform for developing new materials and systems with tailored properties and functionalities.

TECHNOLOGY OVERVIEW

This technology presents a novel, data-driven approach to identifying LLPS-promoting motifs in phase-separating proteins (PhSePs) and designing minimalistic peptides capable of undergoing phase separation and forming functional compartments. The combination of computational analysis and experimental validation offers a powerful method for understanding and manipulating biomolecular condensate formation.

Experimental validation of designed peptides demonstrated that peptide sequences comprising motifs enriched and/or designed from the synergy and co-occurrence of these motifs can undergo LLPS and form compartments. The evaluation of LLPS propensity was conducted under physiological conditions (PBS buffer), with imaging of coacervates using confocal microscopy and Fluorescence Recovery After Photobleaching (FRAP) experiments to assess droplet dynamics.

The technology extends to single-based peptide functional compartments. Peptide sequences that present LLPS-patterns and/or motifs can be combined with functional peptide sequences, allowing for the formation of functional peptide-based coacervates. As an example, a peptide sequence with inherent proficiency in hydrolyzing phosphate ester molecules and binding affinity towards phosphorylated assemblies was designed. This peptide sequence forms biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules (with a 15,000-fold increase in catalytic efficiency) and selectively sequestering phosphorylated proteins.

This technology provides a powerful new approach for understanding the sequence determinants of LLPS and designing minimalistic peptide-based coacervates with enhanced functionality of regulation of substrate recruitment, catalysis mediation and stability. It provides a proof-of-concept for low-complexity, single peptide-based compartments with potential applications in drug delivery, biosensing, and creating synthetic biomolecular condensates for various biotechnological purposes.

This technology has potential application for the detection/recognition of phosphate-specific targets, such as for detecting phosphorylation-dependent supramolecular forms of a protein, such as Tau.

Figure 1: Technology outline – Figure I: Illustration of the strategy used in this work, harnessing liquid-liquid phase separation (LLPS) to enhance peptide catalysis. Depicted are biomolecular coacervates composed of a single peptide (P7), with densely packed peptide domains (where the fully folded ß-hairpin structure is stabilized), and which are proficient at (1) hydrolyzing phosphate ester molecules (e.g., pNPP, p-nitrophenyl phosphate with a 15,000-fold catalytic efficiency increase over soluble peptides; and 2) selectively sequestering phosphoryl assemblies (e.g., BSAp, phosphorylated Bovine Serum Albumin) through affinity interactions.

Figure 2: Enhanced catalytic efficiency and stability of P7 coacervates over time. a Reaction scheme depicting the hydrolysis of p-nitrophenyl phosphate (pNPP) (b) kinetics of P7 peptide-based coacervates (in blue squares) and P7 in bulk solution (red dots) towards the substrate pNPP. The data fitting followed the MichaelisMenten equation V0 = V max½  S =ðKMÞ. Data are presented as mean values ± SD (n = 3) from three independent experiments. In the bulky solution the errors bars are not visible (c) Representative confocal microscopy images showing stability of P7-based coacervates during the hydrolysis of pNPP, at 0 h vs 48 h. Source data are provided as a Source Data file (d), Reaction scheme illustrating the hydrolysis of 2´-[2-benzothiazoyl]−6´-hydroxybenzothiazole phosphate (BBTP). e Representative confocal images showing the progression of BBTP hydrolysis over time. f.Percentage of coacervates that are fluorescent due to the formation of the BBT product during BBTP hydrolysis over time. Data are presented as mean values ± SD (n = 5) of five independent samples. Source data are provided as a Source Data file. All scale bars: 10 μm

FURTHER DETAILS

Calvario J, Antunes D, Cipriano R, Kalafatovic D, Mauša G, Pina, AS*, Investigating Amino acid Enrichments and Patterns in Phase-Separating Proteins: Understanding Biases in Liquid-Liquid Phase Separation. Biomacromolecules 26, 7247 (2025) DOI: https://doi.org/10.1021/acs.biomac.4c00224

Reis DQP, Pereira S, Ramos AP, Pereira PM, Morgado L, Calvário J, Henriques AO, Serrano M, Pina AS* Catalytic peptide-based coacervates for enhanced function through structural organization and substrate specificity. Nature Communications 15, 9368 (2024). DOI: https://doi.org/10.1038/s41467-024-53699-z

STAGE OF DEVELOPMENT

Currently, the technological development is at TRL2.

BENEFITS AND APPLICATIONS

The potential coacervate platform has broad utility as a microreactor, enabling catalysis by tuning sequence‑programmable liquid–liquid phase separation microcompartments that can be used to synthesize intermediates for the pharmaceutical industry in a more sustainable manner. Bioremediation applications include using these compartments to catalyse reactions that require extreme conditions, such as soil decontamination and water purification. Biosensing applications involve sensitive biomarker enrichment to support the development of advanced sensing technologies, owing to their tuneable molecular recognition for diverse targets. The same principles can be exploited for smart therapeutic delivery, in which drugs are encapsulated inside the compartments while the coacervate scaffold functions as the molecular targeting module.

• Single-peptide coacervates: The technology demonstrates the ability to create functional coacervates using a single peptide sequence that can simultaneously induce phase separation, selectively recruit substrates, and mediate catalysis. This simplifies the design of synthetic biomolecular condensates compared to multi-component systems.
• Selective molecular uptake: The coacervates show the ability to selectively sequester phosphorylated proteins over their non-phosphorylated counterparts, demonstrating potential for targeted molecular encapsulation.
• Stability without membranes: Unlike typical coacervates that require surrounding membranes for stability, the peptide-based coacervates demonstrate exceptional stability over extended periods.

INTELLECTUAL PROPERTY

• PCT/IB2025/061103 filed 30.10.2025

OPPORTUNITY

• Available for exclusive and non-exclusive licensing
• Seeking co-development partners and/or Sponsored Research

NOVA Inventors

Ana Pina

Diogo Antunes

Pedro Pereira

Maria Leonor Morgado

Joana Calvário

David Reis