Peptide coacervates, methods, and uses thereof

Abstract

The present disclosure relates to innovative designer peptides for creating single peptide-based coacervates, also known as compartments, liquid-like droplets, or condensates. These designer peptide sequences are inspired by motifs found in disordered regions of intrinsically disordered proteins that undergo liquid-liquid phase separation (LLPS), also known as LLPS-promoting motifs.

Description

D E S C R I P T I O NPEPTIDE COACERVATES, METHODS, AND USES THEREOFTECHN ICAL FIELD

[0001] The present disclosure relates to 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

[0002] Compartmentalization has played a critical role in the emergence of catalysis and in the development of more complex biological systems. For example, it is hypothesized that the first cells emerged from simple lipid vesicles, which provided a protective environment for the chemical reactions that gave rise to the first self-replicating molecules. Membrane confinement, however, is not the only compartmentalization strategy available in nature. Several organisms use liquid-liquid phase separation (LLPS) to concentrate protein and nucleic acid molecules into membrane-less compartments, which enables the organization of cellular functionalities and complex biochemical reactions. LLPS has been shown to promote the nucleation and assembly of actin filaments, and to enhance the enzymatic efficiency of p-carboxysomes and multi-enzyme complexes. These LLPS-derived biomolecular condensates, or coacervates, provide spatial organization and facilitate efficient substrate transfer between enzymes, leading to an overall increase in the efficiency of enzymatic reactions.

[0003] The importance of LLPS in living systems has also fueled its use to create synthetic catalytic systems. Previous studies have used LLPS to confine enzymes in artificial cells via the use of polymers, or to confine substrates in peptide-based condensates. While the impact of phase transitions on enzyme activity is complex and not fully understood, LLPS can create microenvironments that concentrate substrates and enzymes, thus increasing the local substrate concentration and influencing enzyme activity and selectivity.

[0004] Though LLPS has been explored as a strategy to increase the catalytic rates of enzymes in artificial cells, its use for potentiating the activity of catalytic peptides remains unexplored. These catalytic peptides, which are for example widely used as chiral catalysts in organic reactions, present substantial limitations when used in aqueous reactions as their conformational flexibility strongly limits catalytic efficiency. However, the compartmentalization of catalytically active peptides in coacervates presents an enticing avenue to bolster their efficiency by confining their conformational flexibility into more densely packed environments, which may lead to the formation of structured peptide domains with improved catalytic activity. Furthermore, catalytic peptides offer an exciting opportunity as core components ofcoacervates, owing to their remarkable programmability and potential to simultaneously provide structural organization and catalytic functionality. Despite this potential, the systematic investigation of coacervate designs incorporating catalytic peptides that inherently fuse condensate organization with functionality is still largely unexplored. Notably, the encapsulation of the L-dipeptide (Ser-His) within lipid- based vesicles represents the only example of compartmentalization involving catalytic peptides, and this system was mainly explored as a model for the evolutionary competition between protocells.

[0005] 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.

[0006] 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.

[0007] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GEN ERAL DESCRIPTION

[0008] The present disclosure relates to a coacervate, namely peptide-based compartments, liquid-like droplets or condensates, comprising a single peptide with disordered regions that undergo liquid-liquid phase separation (LLPS). This peptide sequence forms biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules and selectively sequestering phosphorylated peptides, stabilizing peptide conformation, and creating a confined environment for peptide catalysis.

[0009] As used herein, the term "coacervate" refers to the dense liquid phase formed by liquid-liquid phase separation of a peptide solution, distinguished from the supernatant (dilute) phase by optical microscopy.

[0010] Surprisingly, the single peptide-based coacervate described in the present disclosure exhibits a 15,000-fold increase in catalytic efficiency for phosphate ester hydrolysis compared to soluble peptides. The present disclosure highlights the potential of a single peptide to induce coacervate formation, selectively recruit substrates, and mediate catalysis, enabling a simple design for low-complexity, single peptide-based compartments with broad implications. Moreover, LLPS emerges as a fundamentalmechanism in the evolution of chemical functions, effectively managing conformational heterogeneity in short peptides and providing valuable insights into the evolution of enzyme activity and catalysis.

[0011] The present disclosure enables selective molecular uptake since the coacervate is has the ability to selectively sequester phosphorylated proteins over their non-phosphorylated counterparts, demonstrating potential for targeted molecular encapsulation. The molecular uptake can lead to enrichment folds of the phosphorylated proteins inside the coacervates of at least of 100-fold. In addition, the peptide-based coacervate demonstrated exceptional stability over extended periods without additional structures.

[0012] An aspect of the present disclosure relates to a coacervate composition comprising a peptide and a suitable solvent; wherein the peptide is represented by the following formula:(A)n-(B)m-(A)p wherein,"A", "B" are independently selected of each other;"A" comprises a phase-separating peptide, wherein "A" is at least 90% identical to a sequence selected from a list consisting of SEQ. ID. 1-701;"B" comprises a peptide for molecular recognition or catalytic activity, wherein "B" is a peptide at least 90% identical to one of the sequences of the list consisting of SEQ. ID. 702-717, SED ID No.764; wherein the peptide comprises up to 50 amino acids; wherein "n" is an integer from 0 to about 50, "m" is an integer from 0 to about 50, "p" is an integer from 0 to about 50; preferably "n" is an integer from 1 to about 25, "m" is an integer from 1 to about 25, "p" is an integer from 1 to about 25; more preferably wherein n=m; and at least one of n, m, p is different of 0.

[0013] In an embodiment, the peptide is at least 90% identical to one of the sequences of the list consisting of SEQ ID No. 1-780, or combinations thereof.

[0014] In an embodiment, the peptide may be at least 95% - 98% identical to a sequence selected from a list consisting of SEQ ID No. 1-780 or combinations thereof; preferably at least 98% - 99% identical; even more preferably identical.

[0015] In an embodiment for better results, the peptide is at least 90% identical to a sequence selected from SED ID No 1, 7, 15, 30, 198, 381, 649, 655, 704, 706, 714, 717, 719, 724 preferably 95% - 98% identical; more preferably at least 98% - 99% identical; even more preferably identical.

[0016] In a preferred embodiment, the peptide is at least 90% identical to a sequence selected from SED ID No 706, 714, 719, 724; preferably 95% - 98% identical; more preferably at least 98% - 99% identical; even more preferably identical.

[0017] The term "phase-separating peptide" refers to a peptide that spontaneously forms a condensed coacervate phase upon dissolution in aqueous buffer under the specified ratio buffer: peptide, peptide concentration, ionic and temperature conditions. The term "peptide for molecular recognition or catalytic activity" refers to a peptide domain that binds or transforms a target molecule, such as a phosphate ester substrate, with measurable affinity or catalytic activity.

[0018] In an embodiment, the coacervate droplet size may range from 1 pm - 50 pm; preferably 1 pm - 10 pm, measured by dynamic laser scattering and confocal microscopy.

[0019] In an embodiment, the zeta potential measured by dynamic light scattering may range from +5mV to +15mV, preferably from +6 mV to +13 mV, at pH 8.0 and 25 °C.

[0020] In an embodiment, the local apparent pH inside the coacervate may range from 5 - 8; preferably 6 - 8; more preferably is 7.

[0021] Herein, the term "local apparent pH" refers to the effective proton activity within a defined region of the coacervate phase, as perceived by molecules or probes located therein. The local apparent pH reflects the microenvironmental proton concentration that may differ from the bulk pH of the surrounding aqueous phase, due to factors such as electrostatic interactions, ion partitioning, charge density, or limited diffusion of ions across the coacervate interface. Accordingly, the local apparent pH represents the pH value that would be measured within the coacervate phase or at its interface, and may be determined indirectly from the protonation state or spectroscopic response of a pH-sensitive compound distributed in the coacervate.

[0022] In an embodiment, the coacervate composition described in the present disclosure may comprise a structured domain wherein the peptide in the structured domain has a folded P-hairpin conformation. Herein, the term "structured domain" refers to a region within the coacervate phase comprising molecules or molecular segments that adopt a defined secondary or tertiary conformation, thereby exhibiting a higher degree of internal organization than the surrounding matrix. In particular embodiments, the structured domain comprises a folded P-hairpin conformation, in which two antiparallel p-strands are connected by a short loop or turn, stabilized by intramolecular hydrogen bonding and, optionally, by hydrophobic or electrostatic interactions. The presence of such structured domains may influence the mechanical stability, local density, or functional properties of the coacervate.

[0023] In an embodiment, the folded P-hairpin conformation was characterized by a circular dichroism spectrum recorded in the wavelength range of 200-300 nm, preferably 210-230 nm, more preferably 215-225 nm. In an embodiment, the folded P-hairpin conformation exhibits a molar ellipticity of -0.25 X105to +0.25 X 105deg.cm2.dmor1at 217 nm and 232 nm, as determined by circular dichroism spectroscopy.

[0024] In an embodiment for better results, the solvent may be an aqueous solvent

[0025] In an embodiment, the volume ratio between the peptide and the solvent ranges from 1:20 (v / v) to 1:5 (v / v). In an embodiment for better results, the volume ratio between the peptide and the solvent may be 1:20 (v / v); preferably 1:10 (v / v); more preferably 1:5 (v / v).

[0026] In an embodiment for better results, the stock concentration of the peptide ranges from 1 mg mL1- 10 mg mL1; preferably from 5 mg mL1- 10 mg mL1.

[0027] The term "suitable solvent" refers to any solvent or solvent mixture that allows the formation of a turbid and not transparent solution of the peptide at the intended concentration and supports phase separation into a coacervate phase under the described conditions. The buffer concentration typically ranges from 0.1 mM to 1000 mM, with pH 7.0 - 8.0, unless stated otherwise.

[0028] In an embodiment, the solvent may be a buffer selected from phosphate buffer, Tris Buffer, HEPES Buffer, MOPS Buffer, PIPES Buffer, MES Buffer, or mixtures thereof.

[0029] In an embodiment, the buffer concentration may range from 10 mM - 200 mM; preferably 50 mM - 150 mM; more preferably 100 mM.

[0030] In an embodiment, the buffer may further comprise a salt; preferably the salt may be selected from: sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCI2), magnesium sulfate (MgS04), sodium phosphate (Na2HPO4or NaH2PO4), ammonium sulfate ((NH4)2SO4), sodium bicarbonate (NaHCO3), potassium nitrate (KNO3), lithium chloride (LiCI), ammonium chloride (NH4CI), sodium acetate (CH3COONa), barium sulfate (BaS04), calcium carbonate (CaCO3), calcium sulfate (CaS04), magnesium chloride (MgCI2), sodium sulfate (Na2SO4), potassium sulfate (K2SO4), sodium citrate (Na3C6H5O7), and sodium fluoride (NaF), or combinations thereof.

[0031] In an embodiment, the salt concentration in the buffer may range from 0.05 M - 2 M; preferably 0.5 M - 1.5 M; more preferably 0.5 M - 1 M.

[0032] In an embodiment , the pH of the coacervate composition may range from 1 - 10; preferably 7 - 9; more preferably 7 - 8, at 27°C measured by absorption, fluorescence spectroscopy and confocal microscopy.

[0033] In an embodiment , the minimum coacervate droplet count per mL ranges from 10 - 100; preferably 50 - 1000; more preferably 100 - 100000, measured by bright-field optical and fluorescent microscopy.

[0034] As used herein, the term "droplet count" denotes the number of discrete coacervate droplets observed per mL under bright-field optical microscopy at 40x magnification. Counts are averaged over ten randomly selected fields.

[0035] In an embodiment, the composition further comprises a nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, fluorophore, analyte-responsive molecule, or combinations thereof. In another embodiment, the analyte-responsive molecule is a chemosensor comprising a pyrene fluorescent reporter and a Zn2+- chelate phosphate-binding group. In a further embodiment, the fluorophore or analyte-responsive molecule has a partitioning efficiency into the coacervate phase between 5% and 90%.

[0036] In an embodiment, the coacervate composition described in the present disclosure may further comprise a substrate.

[0037] As used herein, the term "substrate" refers to any molecule, material, or surface that can interact with, bind to, or be modified by components of the coacervate composition. The substrate may serve as a reactant, a carrier, or a structural support, and can include organic or inorganic compounds, biomolecules (such as peptides, proteins, nucleic acids, lipids, or carbohydrates), or synthetic materials (such as polymers, nanoparticles, or surfaces). The substrate may comprise one or more reactive functional groups capable of participating in covalent or non-covalent interactions.

[0038] In an embodiment, the substrate comprises a reactive functional group selected from phosphate ester, phosphonate, carbonyl, acyl, or amine functional groups.

[0039] In an embodiment, the substrate is selected from: phosphate esters, phosphoric anhydrides, phosphoramidates, phosphonates, nucleoside phosphates, carboxylic acids, esters, amides, or anhydrides, aldehydes, ketones, -hydroxy carbonyl compounds, amino acids or peptides bearing reactive carbonyl or phosphorylated groups, or combinations thereof.

[0040] In an embodiment for better results, the substrate is a phosphate ester substrate or a carbonylcontaining compound.

[0041] In an embodiment, the phosphate ester substrate is selected from: 2'-[2-benzothiazoyl]-6'- hydroxybenzothiazole phosphate (BBTP), p-nitrophenyl phosphate (pNPP), phenyl phosphate, methylumbelliferyl phosphate, fluorescein diphosphate, bis-(p-nitrophenyl) phosphate, 3-O- methylfluorescein phosphate, tyrphostin-phosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine-5'-triphosphate (GTP), diethyl p- nitrophenyl phosphate, or combinations thereof.

[0042] In an embodiment, the carbonyl-containing substrate is selected from: aldehydes, ketones, p- hydroxy aldehydes, p-hydroxy ketones, amino carbonyl compounds, acylated or phosphorylated intermediates thereof, or combinations thereof.

[0043] In an embodiment, the coacervate composition catalyses the hydrolysis of nucleotide phosphates. Upon the hydrolysis of nucleotide phosphates, the coacervates can present emergent behaviours such as mobility with a velocity of at least 0.025 pm / s.

[0044] Another aspect of the present disclosure relates to the use of the disclosed coacervate composition in medicine.

[0045] Even another aspect of the present disclosure relates to the use of the disclosed coacervate composition for the detection / recognition of phosphate-specific targets; preferably for detecting phosphorylation-dependent supramolecular forms of a protein.

[0046] In an embodiment, the protein may be Tau, alpha-synuclein, amyloid precursor protein (APP), MAP2 (microtubule-associated protein 2), neurofilament light chain (NF-L), CRMP2 (collapsin response mediator protein 2), huntingtin protein, TDP-43 (TAR DNA-binding protein 43), p53 (tumor protein 53), GSK-3P (glycogen synthase kinase-3 beta), CDK5 (cyclin-dependent kinase 5), IRS-1 (insulin receptor substrate 1), NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells), elF2a (eukaryotic initiation factor 2 alpha), CREB (cAMP response element-binding protein), among others.

[0047] In an embodiment, the coacervate composition is for use in catalysis or molecular recognition. In a preferred embodiment, the catalytic efficiency (kcat / KM) is at least 5 M1s’1; preferably ranges from 5 M’1s’1- 7 M’1s’1; more preferably 5 M’1s’1- 6 M’1s’1.

[0048] An aspect of the present disclosure relates to the use of the coacervate composition described for the control of the partitioning of guest molecules.

[0049] Another aspect of the present disclosure relates to the use of the disclosed coacervate composition as a catalyst or as a molecular recognition medium.

[0050] In an embodiment, the catalytic efficiency (kcat / KM) was determined by steady-state kinetic analysis of substrate hydrolysis. Reaction rates were measured spectrophotometrically at 25 °C by monitoring the release of p-nitrophenol from p-nitrophenyl phosphate at 405 nm (e = 18 000 M-1cm-1). Michaelis-Menten parameters (K_M and V_max) were obtained by non-linear regression using OriginPro 2024b, and kcat is calculated as V_max / [E]_t, where [E]_t is the total peptide concentration. Catalytic efficiency is reported in M-1s-1.

[0051] The present disclosure also relates to the use of the disclosed coacervate composition in in vitro diagnosis of a disease or condition positively influenced by tau protein; preferably Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy.

[0052] An aspect of the present disclosure relates to a device comprising the described coacervate composition, preferably a biosensor.

[0053] Another aspect of the present disclosure relates to a method for producing the coacervate composition of the present disclosure that may comprise the steps: providing a peptide at least 90% identical to SEQ. ID No. 1-780 or combinations thereof; dissolving the peptide in a solvent forming a peptide solution, preferably an aqueous solvent; inducing liquid-liquid phase separation to form the coacervate composition; and optionally stabilizing the coacervate composition by introducing cross-linking agents.

[0054] In an embodiment, the liquid-liquid phase separation may occur at a temperature of 23°C - 37°C; preferably 1 °C - 35 °C; more preferably 23 °C - 28 °C

[0055] In an embodiment, the inducing step takes from 1 h to 6 h.

[0056] In an embodiment, the method may further comprise a step of entrapping a guest molecule in the coacervate composition.

[0057] In an embodiment, the encapsulation efficiency of the guest molecules in the composition may range from 5% - 100%; preferably 8% - 50%; more preferably 8% - 18%.

[0058] In an embodiment, the encapsulation efficiency (EE%) of guest molecules was determined by separating coacervate droplets from the supernatant via centrifugation (10 000 x g, 10 min, 4 °C). The amount of guest molecule in the coacervate phase (Cencapsuiated) and in the total sample (Ctotal ) was quantified by fluorescence or UV-visible absorbance. Encapsulation efficiency is calculated according to the following equation:

[0059] In an embodiment for better results, the guest molecule is selected from nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, fluorophore or analyte-responsive molecule.

[0060] In one embodiment, a coacervate composition comprising a phase-separating peptide in a coacervate phase achieves selective molecular enrichment such that the efficiency of target molecule uptake, relative to bulk solution, is at least 30-fold, more preferably at least 100-fold, and in some embodiments up to 15,000-fold, when measured for substrate or phosphorylated species partitioning under optimized LLPS conditions. The system demonstrates enhanced selective uptake of phosphorylated biomolecules (such as phosphoproteins) over their non-phosphorylated counterparts, with the enrichment preferably exceeding 100-fold in the chosen assay.

[0061] An aspect of the present disclosure relates to a method for detecting phosphorylated protein level in a biological sample, comprising the following steps: obtaining the disclosed coacervate composition; contacting the biological sample with the coacervate composition, preferably for 3 hours at roomtemperature; imaging the coacervate composition and / or performing spectroscopic analyses (e.g., fluorescence) in the coacervate sample containing the biological sample; and quantifying the presence of protein phosphorylation.

[0062] In an embodiment, the phosphorylated protein is a Tau protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0064] Figure 1: Schematic representation of an embodiment of the present disclosure, harnessing liquid-liquid phase separation (LLPS) to enhance peptide catalysis. Depicted are biomolecular coacervates composed of a single peptide (P7, SEQ. ID No. 706 with densely packed peptide domains (where the fully folded R-hairpin structure is stabilized), and which are proficient at 1) hydrolyzing phosphate ester molecules with a 15,000-fold catalytic efficiency increase over soluble peptides; and 2) selectively sequestering phosphoryl assemblies through affinity interactions.

[0065] Figure 2: Illustration of results of the LLPS of the catalytic peptide P7, which leads to formation of coacervates with structured peptide domains, a, Phase diagrams illustrating the effect of different concentrations of NaCI on the liquid-liquid phase separation (LLPS) of P7, measured by the relative turbidity (100-T%). Different blue shades represent different relative turbidity levels. White circles indicate no LLPS (no relative turbidity), blue circles indicate the presence of coacervates (1 < 100-T% < 10), and the darkest blue indicates the co-existence of peptide aggregates (100-T% > 10). b, Bright-field microscope images depicting peptide-induced coacervation and aggregation at various NaCI and P7 concentrations. Scale bar: 10 pm. c, Conformational analysis of P7 in different states using circular dichroism: soluble with low turbidity (dotted line, stock P7 concentration: 1 mg mL-1), undergoing LLPS (dashed line, stock P7 concentration: 5 mg mL-1), and in the aggregated form (full line, stock P7 concentration: 10 mg mL-1).

[0066] Figure 3: Illustration of results of the partitioning behavior of guest molecules and dynamics of P7 coacervates, a, Confocal microscopy images showing P7 coacervates encapsulating green fluorescent protein (GFP), fluorescein isothiocyanate (FITC), Rhodamine and 4',6'-diamino-2-fenil-indol (DAPI). b, Encapsulation efficiency analysis of the P7 coacervates calculated from fluorescence measurements of bulk solutions and diluted phases. Data are presented as mean values ± SD (n = 3). c, Fluorescence Recovery After Photobleaching (FRAP) analysis of coacervates using ImM FITC. Data are presented as mean values ± SD (n = 3 coacervates), d, Representative confocal images illustrating the FRAP process in an individual coacervate at different time points. All scale bars: 10 pm.

[0067] Figure 4: Illustration of results of the affinity-mediated molecular uptake within P7 coacervates. Time-lapse microscopy captures of the uptake of phosphorylated assemblies inside P7 coacervates. FITC-labelled phosphorylated bovine serum albumin (BSAp) and bovine serum albumin (BSA), Alexa-labeled phosphorylated Tau protein (Taup) and Tau protein, and Alexa-labelled CotBp and CotB were incubated with P7 coacervates for 3h. Representative fluorescence confocal images highlight the molecular uptake within P7 coacervates (a) for FITC-labelled BSAp (c) Alexa-labelled Taup (e) Alexa-labelled CotBp and the exclusion of (b) BSA, (d) Tau, and (f) CotB at the boundaries of P7 coacervates. Scale bar set to 10pm. Full field of view in Figures S8-S12.

[0068] Figure 5: Illustration of results of the 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 (squares) and P7 in bulk solution (dots) towards the substrate pNPP. The data fitting followed the Michaelis-Menten equation Vo = Vmax[S] / (KM). Data are presented as mean values ± SD (n = 3). In the bulky solution the error bars are not visible c, Representative confocal microscopy images showing stability of P7-based coacervates during the hydrolysis of pNPP, at Oh vs 48h. 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). All scale bars: 10pm.

[0069] Figure 6: Illustration of the results of catalytic efficiency of the phase-separating PJ1 peptide (SEQ ID No. 1) with a catalytic triad KYNY (SEQ ID No. 717) of a retro-aldolases (PJ1-KYNY, SEQ ID No 719). a. Reaction scheme depicting Amine catalysis of the retro-aldol reaction, showing the cleavage of the 4- hydroxy-4-(6-methoxy-2-naphthyl)-2-butanon (usually called methodol) into 6- methoxy-2- naphthaldehyde. b, Representative Confocal microscopy images showing PJ1-KYNY coacervates encapsulating FITC. c, Absorbance at 350 nm of the product formation 6- methoxy-2-naphthaldehyde overtime. Data are presented as mean values ± SD (n = 3). All scale bars: 10pm.

[0070] Figure 7: Schematic representation of motifs of the phase-separating peptide construct fused with peptides (e.g., RGGR SEQ ID No 700 or RGGF SEQ ID No 701) and a peptide sequence (EYASD, SEQ ID 715) conferring antimicrobial and autolysin-like catalytic activities. Confocal microscopy images showing the peptide coacervates encapsulating FITC, and FRAP analysis of coacervates using ImM FITC. Data are presented as mean values ± SD (n = 3 coacervates).

[0071] Figure 8: Illustration of the results of catalytic efficiency of the coacervates of a catalytic peptide P29 (SEQ ID No 714) with ATP for ATP hydrolysis, a Confocal microscopy images showing the peptide coacervates encapsulating DAPI, b. Absorbance at 620 nm measuring inorganic phosphate release overtime, quantified using the BIOMOL Green reagent. The assay tracks phosphate generated during ATP hydrolysis, with ADP as the product formed. Data are presented as mean values ± SD (n = 3). c. Trajectories of both coacervates are shown, each starting from the same initial position. Particle velocities were plotted across different cycles. Data analysis and visualization were performed using Fiji (ImageJ).

[0072] Figure 9: Illustration of the results demonstrating the partitioning of a ProxyPhos chemosensor inside P7 coacervates (SEQ. ID No 706) and the detection of phosphorylated molecules, exemplified here by the uptake of phosphorylated BSA into the coacervates with an enrichment of at least 100-fold. Detection is achieved via excimer formation resulting from the proximity of phosphorylated sites in the protein.DETAI LED D ESCRI PTION

[0073] The present disclosure introduces innovative designer peptides for creating single peptide-based coacervates, also known as compartments, liquid-like droplets, or condensates. These designer peptide sequences are inspired by motifs found in the disordered regions of intrinsically disordered proteins that undergo liquid-liquid phase separation (LLPS), also known as LLPS-promoting motifs. The LLPS process modulates the conformational flexibility inherent in peptides, potentially enhancing the functionality of the resulting coacervates, such as improved molecular recognition or enzymatic efficiency

[0074] As used herein, the term "molecular recognition" refers to the specific, reversible, and selective non-covalent interaction between two or more molecules that exhibit complementary structural, electrostatic, hydrophobic, or hydrogen-bonding features. Such interactions enable a host molecule, such as a receptor or sensor, to selectively bind a particular guest molecule or analyte among a mixture of other chemical species.

[0075] As used herein, the term "enzymatic efficiency" refers to the overall effectiveness with which an enzyme catalyses a biochemical reaction under defined conditions and low substrate concentrations, typically expressed as the ratio of the catalytic rate constant (k) to the Michaelis constant (K). This ratio reflects both the turnover rate of the enzyme and its affinity for the substrate.

[0076] As used herein, the term "catalytic efficiency" refers to the ratio between the catalytic turnover rate and the substrate concentration required to achieve that rate. In enzymatic systems, catalytic efficiency corresponds to k / K; in non-enzymatic or artificial catalytic systems, it denotes the rate of substrate conversion per active site relative to substrate availability or other kinetic parameters. The term thus provides a measure of how effectively a catalyst converts substrate into product.

[0077] As used herein, the term "catalytic activity" refers to the rate at which a catalyst promotes a specific chemical reaction under defined experimental conditions. Catalytic activity is quantitatively expressed as the amount of substrate converted to product per unit time per unit mass or mole of catalyst, typically in units such as katal (mol-s-1) or international units (pmol-min_1-mg-1). It reflects the inherent kinetic capability of the catalyst, independent of substrate affinity.

[0078] The present disclosure relates to peptide coacervates, limiting the conformational flexibility of catalytic peptides, which results in structured domains that enhance peptide catalysis.

[0079] In an embodiment, the motif discovery enriched at the disordered regions of phase-separating proteins that undergo LLPS was reached after the analysis of 178 phase-separating proteins, which revealed distinct amino acid enrichment patterns in Droplet Promoting Regions (DPRs) compared to nonDroplet Promoting Regions (NODPRs). Using the "STATITIAN" and "SELECTOR" scripts, using Python 3.11 programming language, Spyder 5.4.3 integrated development environment, and Anaconda Navigator 2.5.2. graphical user interface, 129 significant peptide motifs were identified. These motifs were enriched in Gly, Pro, Ser, Arg, Gin, and Tyr amino acids, suggesting that combinations of polar, hydrophobic, charged, and aromatic residues are crucial for LLPS in small biomolecules. Some motifs showed high repetitiveness within sequences, potentially enhancing multivalent interactions essential for LLPS. The "FREQUENCY" script was used to assess presence and frequency values per DPR sequence. The present disclosure focuses on minimalistic peptide design based on motif discovery and synergy. A computational approach based on the in-house "COMBINER" script was developed to explore and analyse the cooccurrence patterns of motif trios in phase-separating proteins (PhSePs) to design minimalistic peptides capable of undergoing LLPS. This script analyzes peptide motif synergies and physicochemical properties. Ten peptides (PJ1-PJ10 SEQ ID No 1, 7, 15, 30, 198, 381, 649, 655) were designed using both human-driven and computational methods, incorporating diverse motifs and amino acid distributions to test LLPS propensity. The CIDER server was used to analyse physicochemical properties of motif combinations. 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.Example

[0080] In an embodiment, a peptide sequence with inherent proficiency in hydrolyzing phosphate ester molecules and binding affinity towards phosphorylated assemblies was designed.

[0081] In an embodiment, using the flexible catalytic peptide P7 (SEQ ID No. 706) as a model, the formation of reversible biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules and selectively sequestering phosphorylated proteins was induced.

[0082] The following sections provide a detailed description of the materials and methods used in the embodiments of the present disclosure.Trifluoroacetic acid (TFA) Removal of P7 Peptide

[0083] Casio's P7 peptide was subjected to TFA removal using the following procedure: the peptide was dissolved in a 10 mM HCI solution, incubated for 30 minutes, and subsequently lyophilized using aLabConco Freeze-Dryer. This process was repeated twice to ensure efficient removal of TFA. The final TFA content was verified to be below 1%.Coacervation

[0084] The lyophilized powder of P7 peptide was dissolved in 1 mL of distilled water and vortexed until a transparent solution with the desired stock concentration was obtained. The stock concentrations of P7 peptide used were 1 mg mL1, 5 mg mL1, and 10 mg mL1. Coacervation was induced by mixing 60 pL of the P7 stock peptide solution 5 mg mL-1(or 1 mg mL-1and 10 mg mL1) with 240 pL of a 100 mM phosphate buffer solution containing 1 M NaCI at pH 8 (or any other buffer used in the phase diagram) in a 1:5 ratio of peptide to buffer. The samples were incubated for 1 hour at 27°C (±1°C) and then left at room temperature (23°C ± 2°C). For brightfield optical microscopy, 10 pL of the sample was analyzed using a 63x oil immersion lens on a glass slide covered by a functionalized coverslip with 1% Pluronics.Turbidity Measurements

[0085] Turbidity measurements were conducted using a Tecan Infinite M Nano instrument. Absorbance at 600 nm was recorded every 30 seconds for a total duration of 50 minutes. All measurements were carried out at a temperature of 27°C (±1°C). The relative turbidity values reported represent triplicate measurements and were calculated using the formula T relative% = 100 - T% = 100 - [100% x 10 -A600nm]7where A600 is the absorbance at 600 nm. A well containing an equivalent volume of buffer solution served as the blank.Coacervates Imaging

[0086] For imaging samples from the phase diagrams, optical brightfield microscopy was used with a Leica DM6000B upright microscope and for the for the remaining experiments, a Zeiss LSM 880 point laser scanning confocal microscope was used.Cover slides functionalization

[0087] The cover slides were functionalized following the protocol by Pereira, PM et al. Firstly, the cover slides were immersed in absolute ethanol, swirled repeatedly, and then rinsed three times with MilliQ water. This washing procedure was repeated five times. Subsequently, the cover slides were washed with absolute anhydrous acetone, rinsed, and washed three times with MilliQ water. This washing step was also repeated five times. The cover slides were then immersed in 1 M KOH and sonicated for 45 minutes. Afterward, they were rinsed ten times with MilliQ water and immersed in a 1% (w / v) solution of pluronics for 1 hour. Finally, the cover slides were rinsed ten times with MilliQ water to remove any residual solution.Optical Brightfield Microscopy

[0088] For imaging samples from the phase diagrams, a Leica DM6000B upright microscope equipped with an Andor iXon 885 EMCCD camera was used. The MetaMorph V5.8 software was employed to control the microscope, and the images were acquired using a 63x 1.4 NA oil immersion phase-contrast objective (Leica). Image analysis was conducted using ImageJ / FIJLLaser Scanning Confocal Microscopy

[0089] Confocal images were acquired using a Zeiss LSM 880 point scanning confocal microscope equipped with photomultiplier tube detectors (PMTs) and a gallium arsenide phosphide (GaAsP) detector. To visualize individual coacervates, 10 pL of the coacervate mixture was added to Pluronic-functionalized glass slides. Coacervates were imaged using a 63x Plan-Apochromat 1.4 NA DIC oil immersion objective (Zeiss) with laser lines at 405 nm, 488 nm, and 561 nm, and appropriate spectral separation for DAPI, FITC, GFP, and rhodamine§ respectively. The Zeiss Zen 2.3 (black edition) software was used to control the microscope, adjust spectral detection for the excitation / emission of the fluorophores used (following manufactures recommendations). Imaging was performed with 2% laser intensity for all lasers and a gain between 500 and 650. Image analysis was conducted using Zeiss Zen 2.3 (black edition) software and ImageJ / FIJLCircular Dichroism analysis

[0090] CD was employed to investigate the secondary structure of the P7 peptide in different forms: soluble, undergoing liquid-liquid phase separation (LLPS), and as peptide aggregates. The following samples were analyzed: (1) P7 dissolved peptide at a stock concentration of 1 mg mL1in 100 mM sodium phosphate buffer with 1 M NaCI (P7 completely soluble), (2) stock P7 peptide at a concentration of 5 mg mL1in 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0 (undergoing LLPS), and (3) P7 peptide at a stock concentration of 10 mg mL1in 100 mM sodium phosphate buffer at pH 8.0 with 1 M NaCI (peptide aggregates). All samples were prepared following the coacervation protocol. For the CD measurements, quartz cuvettes with a path length of 0.1 cm were used for the P7 peptide stock solution at 1 mg mL1, while cuvettes with a path length of 0.01 cm were used for the 5 mg mL1and 10 mg mL1P7 peptide stock solutions. Far-UV CD spectra in the range of 260 to 205-200 nm were acquired using a J-815 CD spectrometer from Jasco at a temperature of 25°C. The measurements were performed in the appropriate cuvette under a nitrogen flow, with a scanning interval of 1 nm, a spectral bandwidth of 2 nm, and a scanning velocity of 50 nm / min. Each spectrum represents an average of three scans, and buffer signals were subtracted from each scan to obtain the final spectra, and the ellipticity was calculated as described in literature.NMR Samples preparation and experiments

[0091] For NMR studies the P7 peptide samples were prepared according to the coacervation protocol in soluble with low turbidity and undergoing LLPS states. The conditions prepared were P7 peptide in astock concentration of 5mg mL1in lOOmM sodium phosphate buffer with no salt (soluble) and 1 M of NaCI at pH 8.0 (undergoing LLPS) in 92%H2O / 8%D2O. NMR experiments were acquired either at FCT-NOVA or ITQB in Bruker Avance Neo 500 MHz spectrometers equipped with a 5 mM triple resonance Prodigy cryoprobe (TCI). 2D1H TOCSY (40 ms), 2D1H ROESY (250 ms) and 2D1H,13C HSQC spectra were acquired at 23 °C and processed using TopSpin (Bruker Biospin) and analyzed with Poky for assignment. Secondary structure elements were determined with the CSI 3.0 web server.FTIR spectroscopy

[0092] Attenuated total reflection (ATR)-FTIR measurements were performed using a zinc-selenide crystal with a 45° angle of incidence. Samples of P7 peptide were prepared according to the coacervation protocol at three concentrations: 1 mg mL-1in 100 mM sodium phosphate buffer pH 8.0 without salt (soluble), 5 mg mL-1in the same buffer with 1 M NaCI (undergoing LLPS), and 10 mg mL-1in the same buffer with 1 M NaCI. 10 pL of each sample was analyzed. ATR-FTIR spectra were recorded in the amide I band (1550-1720 cm-1) with a spectral resolution of 4 cm-1using a Bruker INVENIO R spectrometer equipped with a DTGS detector. Measurements were conducted at room temperature while purging the sample compartment with dry air. Each spectrum comprised 64 scans, was baseline corrected, and the amide I band was deconvoluted using OriginPro 2024b software.Dynamic Light scattering

[0093] The size of coacervates and their zeta potential was measured by dynamic laser scattering (DLS) (Malvern Zeta-sizer Nano ZS). For size measurements, P7 samples with the different assembly states (soluble with low turbidity, undergoing LLPS and aggregated state) were induced for a final volume of 2mL. Furthermore, 500uL of each sample was transferred to 1.5mL SARSTEDT disposable cuvettes and proceeded to size measurements using scattering angle of 173°, and peptide concentration of 1-5 mg mL-1in 100 mM phosphate buffer IM NaCI (pH 8.0). Lastly, for the measurement of the zeta potential, 700uL from the stock solutions were transferred to ZETASIZER NANO SERIES disposable folded capillary cells. The experiments were performed in triplicates at 25 °C.Partitioning Experiments

[0094] To investigate the encapsulation of guest molecules within the coacervates, partitioning experiments were performed. The guest molecules used were DAPI (4',6-diamidino-2-phenylindole), FITC (3',6'-dihydroxy-6-isothiocyanatospiro[2-benzofuran-3,9'-xanthene]-l-one), GFP (Green Fluorescent Protein), and TMR (tetramethylrhodamine). Firstly, 5 mg of the lyophilized P7 peptide powder was dissolved in 1 mL of distilled water and vortexed until a transparent solution was obtained. Coacervation was induced by mixing 60 pL of the P7 peptide solution with 240 pL of 100 mM phosphate buffer solution containing 1 M NaCI at pH 8.0. This mixture was incubated for 5 minutes at 27°C (±1°C) in the microplate reader INFINITE M NANO+ TECAN, resulting in the formation of milky-colored peptide coacervates. Next,25 pL of the guest molecule solution (ImM fluorophores and 0.1 mM GFP) was added to the coacervate mixture in a 1:12 ratio, resulting in a final volume of 325 pL. Different ratios of 1:5 and 1:20 were also tested. The samples were then incubated for 1 hour at 27°C (±1°C). Then samples of partitioning were centrifuged at room temperature for 30 minutes, and 10 pL of the supernatant was mixed with 190 pL of 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0 for fluorescence measurement. The gain of the guest molecules was measured using the microplate reader INFINITE M NANO+ TECAN. The guest molecule samples were diluted (1:20) into 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0, resulting in a final volume of 200 pL. The percentage of encapsulation (%EE) was calculated using the following equation, where CT represents the total concentration of the fluorophore and Csup represents the concentration in the diluted phase: %EE = (CT-Csup) / CT.Fluorescence recovery after photobleaching (FRAP)

[0095] FRAP experiments were performed on a Zeiss LSM 880 confocal microscope. Sample preparation was performed as described above in the "Confocal Microscopy" section. For fluorescence recovery dynamics assessment, a pre-bleached image of the coacervates was acquired using 488 nm laser line excitation and emission collected with a GaAsP detector. Subsequently, the target coacervates were bleached using the 488 nm laser line at maximum power for 5 seconds. The subsequent recovery of the bleached area was recorded using the same acquisition parameters as the pre-bleached image, with a total recovery time of 87 seconds. The final FRAP recovery curves represent the average of three recovery curves collected from n=3 separate droplets. Correction for photobleaching, normalization, and averaging were performed using ImageJ / FIJI as previously described.Affinity-mediated molecular uptake within P7 coacervates a) Labelling of the phosphorylated and non-phosphorylated proteins: All the proteins were commercially available except for CotBp and CotB, which were produced in Escherichia coli and purified by chromatographic techniques according to previous work. BSAp and BSA were labeled with FITC by using the SIGMA FluoroTagTM FITC conjugation kit according to the supplier's instructions. The sample of FITC-labeled BSAp was quantified using the QuantiProTM BCA Assay Kit, following the manufacturer's instructions. Taup, Tau, CotBp and CotB were fluorescently labelled using the Alexa 488 Microscale Protein Labeling Kit and further quantified also according to the kit supplier's instructions. b) Binding experiments: For the binding experiments, P7 peptide coacervation was induced according to the coacervation protocol, using a lOOmM phosphate buffer solution containing IM NaCI at pH 8.0. After the designated incubation time, the P7 coacervates were transferred to I Bl D I p-Slide 8-well glass bottom previously coated with 1% (w / v) solution of pluronics. A solution of 3pM BSAp-FITC, Taup-Alexa, CotBp- Alexa or lOpM BSA-FITC, Tau-Alexa, CotB-Alexa (controlsamples) was added to the coacervate solution, with a volume of 25pL. The binding experiments were conducted at room temperature for a total of 3 hours. c) Visualization of Binding and Internalization: To observe the binding and internalization of the labeled-phosphorylated and non-phosphorylated proteins into the P7 peptide coacervates, each well of the IBIDI p-Slide 8-well glass bottom, containing the P7 peptide coacervates, was imaged using a Zeiss LSM 800 confocal microscope with Airyscan, equipped with a 63x oil immersion lens and a FITC and Alexa filter. Imaging was performed to capture fluorescence signals indicating the presence of the labeled molecules within the coacervates for a total period of 3 hours. All acquired images were subsequently processed using the ImageJ / FIJI software for further analysis and visualization.Kinetic experiments of P7 coacervates towards the p-nitrophenyl phosphate (pNPP)

[0096] The kinetics of P7 coacervates towards p-nitrophenyl phosphate (pNPP) were investigated using the following protocol. To prepare the P7 coacervates encapsulated with pNPP, a final volume of 1 mL was used, following the partitioning protocol. The peptide stock solution of 5 mg mL1and the coacervation condition of 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0 were utilized. In a cuvette, 186.4 pL of the 5 mg mL1P7 peptide solution was mixed with 738.46 pL of buffer and incubated for 5 minutes at 27°C (±1°C) to induce coacervation. Subsequently, 76.923 pL of pNPP substrate at different concentrations (1, 5, 10, 12, 18 mM) were added to a final volume of 1 mL, maintaining the ratios specified in the partitioning protocol. The samples were then incubated for 1 hour at 27°C (±1°C). The formation of p-nitrophenol (pNP) was monitored by measuring the absorbance at 405 nm. The analysis of kinetic experiment data conducted with P7 coacervates involved two key corrections: (1) to account for the turbidity interference caused by coacervates, the absorbances of P7 coacervates without pNPP were subtracted from the experimental readings, and (2) to account for the autohydrolysis of the substrate in bulk solution, the absorbances of pNPP over time (without coacervates) were also subtracted from the experimental readings. These corrections allowed for a more accurate assessment of the catalytic activity of P7 coacervates by isolating the pNPP hydrolysis specifically caused by the peptide-based coacervates. The initial velocity rate of each substrate concentration catalyzed by the P7 coacervates was determined by analyzing the linear phase of pNP formation over time (90 minutes). The kinetic parameters, namely Vmax and KM, were determined using OriginPro9 software by fitting the data to the Michaelis-Menten equation V0 = Vmax[S] / (KM). The rate of catalysis was calculated using the equation Rate (s-1) = [Product formed] (M) / ([P7] (M) x time (s)). The experiments were performed in triplicates, and the obtained kinetic parameters were compared with those reported by Pina et al.19for P7 in bulky solution under optimal conditions.Kinetic experiments of P7 coacervates towards the 2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate (BBTP)

[0097] For the kinetic experiments of P7 coacervates with BBTP substrate, the commercially available AttoPhos® AP Fluorescent Substrate System from Promega was used. A 1 mM solution of AttoPhos was prepared by dissolving the contents of the attophos vial (36 mg) in 60 mL of AttoPhos® Buffer. To prepare the P7 coacervates encapsulated with BBTP substrate, the partitioning protocol was followed with a final volume of 1 mL. The peptide stock solution of 5 mg mL-1 and the coacervation condition of 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0 were utilized. In a cuvette, 186.4 pL of the 5 mg mL-1 P7 peptide solution was mixed with 738.46 pL of 100 mM sodium phosphate buffer with 1 M NaCI at pH 8.0, followed by incubation for 5 minutes at 27°C (±1°C) to induce coacervation. Subsequently, 76.923 pL of the BBTP substrate at a concentration of 1 mM was added, and the mixture was further incubated for 1 hour at 27°C (±1°C). The formation of the alcohol product, 2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole (BBT anion), was monitored after 5 days (120 hours) using confocal microscopy with the FITC filter. The fluorescence gain was set to 650, and the PMT gain was set to 500. The laser intensity was maintained at 2.0 for all experiments.

[0098] All acquired images were processed using the I mageJ / F IJ I software for analysis and quantification.

[0099] The following sections provide a detailed description of the results of the embodiments of the present disclosure.LLPS induced reversible self-coacervation of peptides with conformational flexibility.

[0100] The catalytic peptide P7 (KVYFSIPWRVPM-NH2, SEQ ID No. 706) has a primary sequence rich in residues recognized for their role in triggering LLPS, including R, K, S and P. Therefore, P7 is ideally placed to test that catalytic peptides can be used as a single component that merges functionality and structural organization within coacervates, through LLPS. To test this, first, a systematic high-throughput approach was performed, screening how different NaCI and peptide concentrations influence P7 coacervation. The resulting samples were characterized using relative turbidity measurements and optical brightfield microscopy (Fig. 2).

[0101] In all the sequences from SEQ. ID 1-780 the organism's name is "synthetic construct", molecule type is "amino acid", qualifier molecule type is "protein", as shown for SEQ. ID 502 in the next table:

[0102] In an embodiment, the turbidity values consistently increased with increasing concentrations of peptide and NaCI (Fig. 2a). For each peptide concentration tested, turbidity also increased as the NaCI concentration increased, suggesting peptide-driven coacervation (Figs. 2a-2b). The presence ofcoacervates was confirmed by optical brightfield microscopy, observing increased abundance of coacervate with IM NaCI and at peptide concentrations from 1 to 5 mg mL1, with the highest coacervate formation observed at 5 mg mL1(Fig. 2b). Interestingly, higher peptide concentrations (10 mg mL1) led to the formation of both coacervates and peptide aggregates (Fig 2a-2b). At this high peptide concentration, increasing NaCI concentrations increased the quantity and size of aggregates but did not result in coacervates formation. These findings highlight the intricate interplay between coacervation and aggregation, with the peptide and salt concentrations serving as key regulators. The concentration of salts plays a crucial role in driving the phase separation process, underscoring the significant contribution of hydrophobic interactions to coacervate formation, which are enhanced in the presence of salt. However, it is important to emphasize that, at the highest concentrations of peptides and salts, the prevailing salting-out effect leads to the precipitation of peptides. According to the test of the effect of the environmental conditions in the aggregate to coacervate equilibrium, when the aggregate rich solution is diluted to lower the peptide concentration from 10 to 5 mg mL1(while retaining 1.00 M NaCI), the disappearance of aggregates and coacervate formation its observed, highlighting the reversible nature of coacervation.

[0103] In an embodiment for, the impact of temperature on P7 coacervation under optimal conditions (stock peptide concentration of 5 mg mL1in 100 mM sodium phosphate buffer, 1.00 M NaCI, pH 8), using turbidity measurements and optical brightfield microscopy. Different temperatures ranging from 23°C to 37°C. Coacervate formation was observed within one hour of incubation, with temperatures above 27°C showing higher turbidity values, favoring coacervate formation compared to temperatures below 25°C. These data are consistent with previous studies on similar systems where coacervation is primarily driven by hydrophobic interactions, which are known to be temperature-sensitive. Similar to the effects of higher peptide concentrations, higher coacervation temperatures (>35°C) also led to aggregation.

[0104] In an embodiment, the temporal dynamics of the coacervation process through time-course analysis was characterized. The samples were initially incubated at 27°C to induce coacervation and subsequently maintained at room temperature (23°C ± 2°C) for a total duration of 6 hours. This analysis revealed that while the coacervates had a larger size at one-hour coacervation, their size gradually decreased over time due to the formation of liquid bridges, leading to an increased number of coacervates. This phenomenon is characteristic of liquid-like condensates, which can fuse, deform or, as in this case, split into two coacervates. This cooling process from 27°C to room temperature seems to impact coacervate dynamics. These temperature-dependent changes appear to contribute significantly to the observed division and size decrease of the coacervates. The observed coacervate size range was consistent with values reported in the literature, typically 1-10 pm. This size range of the coacervates also corroborated by dynamic light scattering (DLS). Having demonstrated that P7 can form coacervates, circular dichroism (CD) analysis was used to examine the influence of LLPS on the conformationalconstraints of this flexible catalytic peptide during phase separation. The secondary structure of the peptide was analyzed in three distinct assembly states: soluble (any of the conditions 1 mg mL1with no salt, 5 mg mL1with no salt or 1 mg mL1with IM NaCI showing absence or low turbidity), undergoing LLPS (5 mg mL1IM NaCI), and in the aggregate (10 mg ml1IM NaCI). The LLPS state of the peptide exhibits a CD signature, with a minimum ellipticity observed around 213-219 nm and a maximum at 232 nm (Fig. 2c), characteristic of a fully folded P-hairpin conformation (typified by a broad negative band at approximately 217 nm). These results suggest that LLPS induces the stabilization of the secondary structure of P7, transforming a flexible hairpin-like peptide in solution into a fully folded P-hairpin structure in the LLPS state. The CD analysis also shows that in the aggregated state, the peptide loses its conformational order. Further supporting this conformational equilibrium, NMR analysis for the soluble and undergoing LLPS assembly states corroborate these findings. The secondary structures propensities calculated from the assigned chemical shifts revealed a beta-hairpin in LLPS state compared with a flexible hairpin-like peptide structure in the soluble state as showed by the presence of a turn within two random coil stretches.

[0105] In an embodiment, Fourier-transform infrared (FTIR) spectroscopy was employed as a complementary method to identify the secondary structure of the P7 peptide in various assembly states. The FTIR spectra, reveal distinct differences in the amide I band region, indicative of secondary structure variations. Deconvolution of the amide I band in the soluble state primarily indicates the presence of a random coil conformation (band at 1640 cm1), with a minor band at 1685 cm'1attributed to a p-turn, reinforcing the presence of a hairpin-like peptide structure.

[0106] In an embodiment, in the P7 coacervates, the p-sheet conformation was significantly higher, accounting for approximately 70% of the structure. This was evidenced by the contributions of p-sheet bands at 1623 cm'1and 1663 cm1, along with a p-turn band at 1683 cm1, thus reinforcing the presence of a P-hairpin structure in the LLPS state. Conversely, in the aggregated assembly state, there is a significant decrease in p-sheet content (down to 20%) compared to the P7 coacervates, with a more pronounced random coil conformation, corroborating a loss of conformational order. These FTIR findings are consistent with the results obtained from CD and NMR analyses, indicating an enhanced content of p- sheet and higher structural organization in P7 coacervates compared to the soluble and aggregated states. Collectively, these structural investigations underscore the critical role of LLPS in modulating the conformational flexibility of the P7 peptide, stabilizing structured domains. These findings also underscore the delicate interplay between LLPS and aggregation, which is governed by the environmental conditions utilized to induce LLPS and is contingent upon the P-hairpin conformation. Thus, overall, these findings demonstrate that P7 undergoes a transition from a flexible hairpin-like peptide to a coacervate-forming peptide with a structured domain exhibiting a fully folded P-hairpin structure, highlighting the potential importance of such structural plasticity in the evolution of chemical function.

[0107] In an embodiment, the coacervate compartment is capable of partitioning or encapsulating any fluorophore or analyte-responsive molecule, such as chemosensors containing pyrene (as a fluorescent reporter) and a Zn2+-chelate (as a phosphate-binding group), but also compounds that identify and quantify specific chemical substances by transducing target analyte interactions into analytically useful signals. In an embodiment, partitioning efficiencies range between 5% and 90%. This encapsulation enables the fabrication of biosensor devices for a broad range of applications and may be extended to the detection and measurement of any biomolecule using the same molecular recognition and signal transduction concept.

[0108] In an embodiment, P7 coacervates selectively controlled the partitioning of guest molecules. Coacervates are dynamic compartments, with the capacity to partake in high internal mixing and rapid exchange with the external environment. Thus, the potential to sequester and partition specific molecules inside P7 coacervates was explored. First, the concentration ability of the P7-based coacervates for the aromatic dye molecules (9-(2-Carboxyphenyl)-6-(diethylamino)-N,N-diethyl-3H-xanthen-3-iminium chloride (rhodamine), Fluorescein 5-isothiocyanate (FITC), and 4',6-diamidino-2-phenylindole (DAPI)) and for Green Fluorescent Protein (GFP) was investigated. The partitioning of these molecules under optimized conditions was assessed, the encapsulation efficiency was quantified (%EE = (c solution - c diluted phase) / cSoiution) and the resulting coacervates images were captured by laser scanning confocal microscopy (Fig- 3).

[0109] In an embodiment, the P7 coacervates presented a net positive surface charge of +9.4 ± 3.2 mV as determined by dynamic light scattering (DLS) measurements at pH 8.0. Local apparent pH inside coacervate droplets and the dilute phase was measured using 5-(and 6)-carboxy SNARF-1 (SNARF), a pH- sensitive indicator dye. By partitioning SNARF-1, it was possible to measure the local apparent pH of both the coacervate phase and the dilute phase, which were estimated to be approximately 7.3 and 7.4, respectively. The slightly positive charge of P7 is predicted to promote the segregation of negatively charged molecules inside the coacervates through electrostatic interactions. Indeed, the negatively charged molecule GFP exhibited a higher encapsulation efficiency than neutral dye molecules (Fig. 3a-3b). For neutral dyes, hydrophobicity seems to be a dominant factor affecting partitioning, with encapsulation efficiency increasing as hydrophobicity decreases. Less hydrophobic molecules (with a lower octanolwater partition coefficient, logP), such as FITC and DAPI, preferentially partition into the coacervates, while dyes with a higher hydrophobic character (rhodamine) accumulate at the coacervate boundaries (Fig. 3a). Different ratios of the peptide P7-to-guest molecule ratio were used. These findings revealed that for hydrophobic molecules such as FITC and Rhodamine, the partitioning is largely independent of concentration, suggesting that hydrophobic interactions, governed by the P7 peptide sequence, play a dominant role. For less hydrophobic molecules like DAPI, the partitioning efficiency increases with higherpeptide-to-guest molecule ratios, indicating that additional interactions, such as, for example, hydrogen bonding, are involved.

[0110] In an embodiment, fluorescence recovery after photobleaching (FRAP) was applied to characterize the dynamics of coacervates, with FITC as a representative guest molecule (Fig. 3c-3d). The results demonstrated a significant (~60%) fluorescence recovery in the coacervates, affirming their liquidlike nature.

[0111] Collectively, these observations demonstrated that P7 coacervates selectively control the dynamic partitioning of guest molecule and suggest that n-n interactions between the aromatic groups of the dye molecules and the aromatic residues of the P7 peptide together with hydrogen bonding between the peptide and less hydrophobic guest molecules mediate partitioning within the coacervates. Therefore, the functional properties of peptide coacervates are intricately tied to the encoded peptide sequence, which influences the partitioning of molecules within the coacervates.

[0112] In an embodiment, P7 coacervates selectively recruited phosphorylated molecules. The capability of peptide-based coacervates to selectively recruit and uptake phosphorylated molecules in comparison to their non-phosphorylated counterparts was explored. P7 peptide exhibits a dissociation constant in the micromolar range specifically towards phosphorylated assemblies. Leveraging this property, the P7 coacervates were incubated with different fluorophore-labelled phosphorylated proteins and their nonphosphorylated counterparts to explore the applicability of the P7 coacervates. Three distinct proteins were evaluated: phosphorylated bovine serum albumin (BSAp), its non-phosphorylated counterpart (BSA), phosphorylated human microtubule-associated Tau protein (Taup) along with its non- phosphorylated form (Tau), and Bacillus subtilis spore surface protein B (CotB) and its phosphorylated variant (CotBp). BSA is a versatile model protein to study, suitable for a broad range of applications. Tau is an intrinsically disordered protein vital to neuronal structure, which undergoes a pathological transformation when excessively phosphorylated, leading to brain dysfunction. CotB is an abundant bacterial spore protein that is hyperphosphorylated by a kinase, CotH in the presence of a facilitator protein, CotG, the three proteins forming a phosphorylation module that patterns the spore surface layers. BSA, Tau, and all their phosphorylated versions are intrinsically negative due to their isoelectric points and degrees of phosphorylation. In contrast, CotB is intrinsically positive.The P7 coacervates were incubated with FITC / Alexa-labeled phosphorylated proteins or FITC / Alexa-labeled non-phosphorylated proteins, monitoring their uptake by confocal microscopy over a 3-hour period. Interestingly, it was observed coacervate internalization of BSAp / Taup / CotBp (Fig. 4), but not of non-phosphorylated BSA, Tau and CotB which is excluded and remains confined to the boundaries of the coacervates even after the 3-hour period (Fig. 4). This selective uptake is particularly noteworthy given that BSA and Tau are intrinsically negatively charged proteins, which might be expected to interact electrostatically with the positively charged coacervate surface. These findings strongly suggested that electrostatic interactionsare not the primary driving force for protein sequestration within the coacervates. Instead, these results indicate that the selective binding properties of the P7 peptide sequence towards phosphorylated assemblies play a crucial role in the uptake mechanism, as demonstrated by the preferential uptake of phosphorylated proteins over their non-phosphorylated counterpart. This phosphate-specific recognition suggests that it may be possible to incorporate other molecular recognition capabilities towards specific targets within the sequences of phase-separating peptides, paving the way for the creation of a novel class of functional biomolecular coacervates capable of mediating selective molecular uptake, resembling the processes observed in cellular functions. In an embodiment, this discriminative capacity of the P7- coacervates of selectively detecting phosphorylation levels in Tau over non-phosphorylated Tau holds promise for future diagnostic tools. The detection of phosphorylation in tauopathies is paramount for diagnosis, comprehending disease mechanisms, and developing treatments. Detection of phosphorylated CotB is a proxy for proper functioning of the CotB / CotG / CotH phosphorlytaion module and thus for the proper structural organization of the spore surface layers, which in turn is important for the environmental persistence of spores and their interaction with host cells and abiotic surfaces.

[0113] In an embodiment, the peptide-based coacervates evidenced enhanced catalytic efficiency. Having shown that LLPS can induce the formation of P7 coacervates and lead to the emergence of structured domains within the coacervates, then the impact of constraining peptide conformation through LLPS on P7 catalytic efficiency was explored. First, a standard colorimetric phosphate ester hydrolysis reaction employing p-nitrophenyl phosphate (pNPP) as the substrate was used (Fig. 5a). The catalytic activity of P7 under LLPS conditions (5 mg mL’1stock concentration peptide 100 mM sodium phosphate pH 8.0, 1 M NaCI) was assessed, both with and without triggering coacervate formation. To induce coacervation, the peptide and buffer solution at a ratio of 1:5 (peptide:buffer) and incubated the mixture at 27°C. This resulted in the formation of coacervates. Subsequently, the substrate into the coacervation phase was introduced at 27°C to initiate the catalytic reaction. To better estimate pNPP sequestration within the coacervates, the encapsulation efficiency of pNPP was determined, showing an efficiency ranging from 8% - 18%, depending on the pNPP concentration. These results indicated that pNPP sequestration inside the coacervates is not significantly higher than in the bulk solution. In the condition without triggering coacervation, the peptide was dissolved in the same buffer solution to achieve the same final concentration of 1 mg mL’1. This concentration was selected to correspond with the final peptide concentration in the coacervate samples, while maintaining the peptide in a soluble state and preventing phase separation. The substrate was then added to the resulting solution, and the catalytic reaction was also conducted at 27°C (Fig. 5b). Without coacervation, no observable hydrolysis of pNPP occurred even after 90 hours. However, under LLPS conditions, the initial rate of the reaction displayed substrate concentration dependency consistent with the Michaelis-Menten model (Fig. 5b). The catalytic parameters obtained were: kcat of (4.9 ± 0.6) x 10'3s’1, KMof (8.2 ± 3.2) x 10’4M and a catalytic efficiency, kcat / KM, of 5.9 ± 0.2 (Fig. 5b, Table 1). Additionally, the stability of the coacervates was evident even after48 hours of incubation (Fig. 5c). The comparison between the kinetic parameters obtained with P7 peptide-based coacervates to those previously determined for the peptide P7 in a bulk solution under catalytic optimal conditions (i.e., P7 final concentration of 50pM in 100 mM sodium phosphate pH 8.0 with no salt, and catalytic reaction temperature 25 °C) , show that the coacervates exhibit a 20-fold decrease in KM, a 500-fold increase in kcat, and a 15,000-fold improvement in catalytic efficiency (Table 1). Thus, catalysis is substantially improved in peptide-based coacervates.Table 1 -Kinetic parameters of the peptide P7 under bulky solution and upon LLPS.

[0114] In an embodiment, to confirm that the phosphate ester hydrolysis reaction occurred within the P7 coacervates a different substrate, 2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate (BBTP), was employed which, upon cleavage by a phosphatase biocatalyst, produces inorganic phosphate and the fluorescent alcohol, 2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole (BBT) (Fig. 5d). Analysis of this reaction using confocal microscopy showed that the number of coacervates exhibiting fluorescence increases over time, reaching a maximum after 120 hours of reaction, further highlighting the ability of peptide-based coacervates to hydrolyze phosphate ester molecules (Figs. 5e-5f). Remarkably, these coacervates remain stably dispersed in the catalytic environment for the entire 120-hours duration of the reaction (Figs. 5e).

[0115] Collectively, these data demonstrate that LLPS-mediated compartmentalization can enhance the catalytic efficiency of P7, as a result of the conformational stabilization of the peptide within coacervates. Through precise control of peptide conformation via LLPS, catalytic efficiencies that rival those observed in phosphatase enzymes were achieved. Furthermore, unlike typical coacervates that often require surrounding membrane layers to prevent droplet coalescence and maintain stability for extended periods, the P7 peptide-based coacervates, an embodiment of the present demonstrated exceptional stability without the need for such additional structures. These results demonstrate that peptide-based coacervates can not only serve as efficient microreactors but also undergo in situ conversions into condensed structures with improved stability.

[0116] In an embodiment, a phase-separating peptide (PJ1: FGGGRGGFGGDRGG, SEQ ID No 1) fused to a catalytic triad motif of an enzyme KYNY (SEQ ID No. 717) comprising the sequence FGGGRGGFGGDRGGKYNY (SEQ ID No 719) is capable of undergoing phase separation in aqueous solvents,thereby facilitating the selective partitioning of fluorophores, such as but not limited to fluorescein isothiocyanate, and other molecular species, into the coacervate phase. This construct demonstrates enhanced catalytic efficiency compared to non-phase-separating analogues in aqueous environments. Fig.6 depicts the results of catalytic efficiency of the phase-separating PJ1 peptide (SEQ ID No 1) with a catalytic triad KYNY (SEQ ID No 717) of a retro-aldolases (PJ1-KYNY, SEQ ID 719).

[0117] In another embodiment, a phase-separating peptide fused with peptides (e.g., RGGR or RGGF) and a peptide sequence (EYASD) possessing antimicrobial and autolysin-like catalytic activities is capable of undergoing phase separation. This allows for the partitioning of fluorophores, including but not limited to fluorescein isothiocyanate, as well as other molecular species, into the coacervate phase. Fig 7 depicts the results obtained with a phase-separating peptide construct fused with peptides (e.g., RGGR SEQ ID No 700 or RGGF SEQ ID No 701) and a peptide sequence (EYASD, SEQ ID No 715) comprising the sequence RGGREYASDRGGF (SEQ ID No 724) conferring antimicrobial and autolysin-like catalytic activities.

[0118] As used in the specification and claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a sample" includes a plurality of samples, including mixtures thereof.

[0119] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0120] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0121] As used herein, the terms "pharmaceutically acceptable" and "cosmetically acceptable" are used interchangeably and refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. More specifically, pharmaceutically acceptable refers to a material, compound, or composition which is suitable for use in contact with the skin, scalp, or hair. Pharmaceutically acceptable materials are known to those of ordinary skill in the art.

[0122] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unlessthe context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0123] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The abovedescribed embodiments are combinable.

[0124] The following dependent claims further set out particular embodiments of the disclosure.Sequence list:

Claims

C L A I M S1. Coacervate composition comprising a peptide and a suitable solvent, wherein the peptide is represented by the following formula:(A)n-(B)m-(A)p wherein,"A", "B" are independently selected of each other;"A" comprises a phase-separating peptide, wherein "A" is at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 1-701;"B" comprises a peptide for molecular recognition or catalytic activity, wherein "B" is a peptide at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 702-717, SED ID No. 764; wherein the peptide comprises up to 50 amino acids; wherein "n" is an integer from 0 to about 50, "m" is an integer from 0 to about 50, "p" is an integer from 0 to about 50; preferably wherein "n" is an integer from 1 to about 25, "m" is an integer from 1 to about 25, "p" is an integer from 1 to about 25; more preferably wherein n=m; at least one of n, m, p is different of 0.

2. Coacervate composition according to the previous claim, wherein the peptide comprises a sequence at least 90% identical to a sequence selected from a list consisting of SEQ ID No. 1-780, or combinations thereof.

3. Coacervate composition according to any of the previous claims, wherein the peptide comprises a sequence at least 95% - 98% identical to a sequence selected from a list consisting of SEQ. ID. 1-780, or combinations thereof; more preferably at least 98% - 99% identical; even more preferably identical.

4. Coacervate composition according to any of the previous claims comprising a peptide at least 90% identical to a sequence selected from SED ID No. 1, 7, 15, 30, 198, 381, 649, 655, 704, 706, 714, 717, 719, 724 preferably 95% - 98% identical; more preferably at least 98% - 99% identical; even more preferably identical.

5. Coacervate composition according to any of the previous claims, wherein the coacervate droplet size ranges from 1 pm - 50 pm; preferably 1 pm - 10 pm.

6. Coacervate composition according to any of the previous claims, wherein the zeta potential of the coacervates, measured by dynamic light scattering, ranges from +5mV to +15mV, preferably from +6 mV to +13 mV, at pH 8.0 and 25 °C. .

7. Coacervate composition according to any of the previous claims, comprising a structured domain wherein the peptide in the structured domain has a folded P-hairpin conformation.

8. Coacervate composition according to the previous claim, wherein the folded P-hairpin conformation exhibits a molar ellipticity of -0.25 X 105to +0.25 X 105deg.cm2.dmol-1measured by circular dichroism spectroscopy in a wavelength ranging from 200 nm - 300 nm; preferably 210 nm - 230 nm; more preferably 215 - 225 nm.

9. Coacervate composition according to any of the previous claims, wherein the solvent is an aqueous solvent.

10. Coacervate composition according to any of the previous claims, wherein the volume ratio between the peptide and the solvent ranges from 1:20 (v / v) to 1:5 (v / v).

11. Coacervate composition according to any of the previous claims, wherein stock concentration of the peptide ranges from 1 mg mL1- 10 mg mL1; preferably from 5 mg mL1- 10 mg mL1.

12. Coacervate composition according to any of the previous claims, wherein the solvent is a buffer selected from phosphate buffer, Tris Buffer, HEPES Buffer, MOPS Buffer, PIPES Buffer, MES Buffer, or mixtures thereof.

13. Coacervate composition according to the previous claim, wherein the buffer concentration ranges from 10 mM - 200 mM; preferably 50 mM - 150 mM; more preferably 100 mM.

14. Coacervate composition according to any of the previous claims 12-13, wherein the buffer further comprises a salt; preferably the salt is selected from sodium chloride (NaCI), potassium chloride (KCI), calcium chloride (CaCI2), magnesium sulfate (MgS04), sodium phosphate (Na2HPO4or NaH2PO4), ammonium sulfate ((NH4)2SO4), sodium bicarbonate (NaHCO3), potassium nitrate (KNO3), lithium chloride (LiCI), ammonium chloride (NH4CI), sodium acetate (CH3COONa), barium sulfate (BaS04), calcium carbonate (CaCO3), calcium sulfate (CaS04), magnesium chloride (MgCI2), sodium sulfate (Na2SO4), potassium sulfate (K2SO4), sodium citrate (Na3C6H5O7), and sodium fluoride (NaF), or combinations thereof.

15. Coacervate composition according to the previous claim, wherein the salt concentration in the buffer ranges from 0.05 M - 2 M; preferably 0.5 M - 1.5 M; more preferably 0.5 M - 1 M.

16. Coacervate composition according to any of the previous claims, wherein the pH of the coacervate composition ranges from 1 - 10; preferably 7 - 9; more preferably 7 - 8.

17. Coacervate composition according to any of the previous claims, wherein the minimum coacervate droplet count per mL ranges from 10 - 100; preferably 50 - 1000; more preferably 100 - 100000.

18. Coacervate composition according to any of the previous claims further comprising nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, a fluorophore, analyte-responsive molecule, or combinations thereof.

19. Coacervate composition according to the previous claim wherein the analyte-responsive molecule is a chemosensor comprising a pyrene fluorescent reporter and a Zn2+-chelate phosphate-binding group.

20. Coacervate composition according to any of the previous claims 19-20 wherein the nucleic acids, lipids or lipid-like molecules, carbohydrates, protein, peptide, organometallic molecules, organic molecules, inorganic molecules, fluorophore, analyte-responsive molecule, or combinations thereof has a partitioning efficiency into the coacervate phase between 5% and 90%.

21. Coacervate composition according to any of the previous claims further comprising a substrate.

22. Coacervate composition according to the previous claim, wherein the substrate comprises a reactive functional group selected from phosphate ester, phosphonate, carbonyl, acyl, or amine functional groups.

23. Coacervate composition according to any of the previous claims 22-23 wherein the substrate is selected from: phosphate esters, phosphoric anhydrides, phosphoramidates, phosphonates, nucleoside phosphates, carboxylic acids, esters, amides, or anhydrides, aldehydes, ketones, - hydroxy carbonyl compounds, amino acids or peptides bearing reactive carbonyl or phosphorylated groups, or combinations thereof.

24. Coacervate composition according to any of the previous claims 22-24 wherein the substrate is a phosphate ester substrate or a carbonyl-containing compound.

25. Coacervate composition according to the previous claim wherein the phosphate ester substrate is selected from: 2'-[2-benzothiazoyl]-6'-hydroxybenzothiazole phosphate (BBTP), p-nitrophenyl phosphate (pNPP), phenyl phosphate, methylumbelliferyl phosphate, fluorescein diphosphate, bis- (p-nitrophenyl) phosphate, 3-O-methylfluorescein phosphate, tyrphostin-phosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine-51- triphosphate (GTP), diethyl p-nitrophenyl phosphate, or combinations thereof.

26. Coacervate composition according to the previous claim 25 wherein the carbonyl-containing substrate is selected from: aldehydes, ketones, p-hydroxy aldehydes, p-hydroxy ketones, amino carbonyl compounds, acylated or phosphorylated intermediates thereof, or combinations thereof.

27. Coacervate composition according to any of the previous claims for the detection / recognition of phosphate-specific targets; preferably for detecting phosphorylation-dependent supramolecular forms of a protein.

28. Coacervate composition according to the previous claim, wherein the protein is Tau, alpha-synuclein, amyloid precursor protein (APP), MAP2 (microtubule-associated protein 2), neurofilament light chain (NF-L), CRMP2 (collapsin response mediator protein 2), huntingtin protein, TDP-43 (TAR DNA-binding protein 43), p53 (tumor protein 53), GSK-3P (glycogen synthase kinase-3 beta), CDK5 (cyclin- dependent kinase 5), IRS-1 (insulin receptor substrate 1), NF-kB (nuclear factor kappa-light-chain- enhancer of activated B cells), elF2a (eukaryotic initiation factor 2 alpha), CREB (cAMP response element-binding protein), or combinations thereof.

29. Coacervate composition according to any of the previous claims for use in catalysis or molecular recognition.

30. Coacervate composition according to the previous claim, wherein the catalytic efficiency (kcat / KM) is at least 5 M1s’1; preferably ranges from 5 M’1s’1- 7 M’1s’1; more preferably 5 M’1s’1- 6 M’1s’1.

31. Use of a coacervate composition as described in any of the previous claims 1-28 in in vitro diagnosis of a disease or condition positively influenced by tau protein; preferably Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, or chronic traumatic encephalopathy.

32. Device comprising the coacervate composition according to any of the previous claims, preferably a biosensor.

33. Method for producing the coacervate composition, comprising the following steps: providing a peptide at least 90% identical to SEQ. ID No. 1-780, or combinations thereof; dissolving the peptide in a solvent forming a peptide solution, preferably an aqueous solvent; inducing liquid-liquid phase separation to form the coacervate composition; and optionally stabilizing the coacervate composition by introducing a cross-linking agent.

34. Method for producing the coacervate composition according to the previous claim, wherein the liquid-liquid phase separation occurs at a temperature of 23°C - 37°C; preferably 27 °C - 35 °C; more preferably 23 °C - 28 °C.

35. Method for producing the coacervate composition according to any of the previous claims 33-34, wherein the inducing step takes from 1 h to 6 h.

36. Method for producing the coacervate composition according to any of the previous claims 33-35 further comprising a step of entrapping a guest molecule the coacervate composition, preferably in the coacervate phase.

37. Method for producing the coacervate according to the previous claim, wherein the encapsulation efficiency of the guest molecule ranges from 5% - 100 %; preferably 8% - 50%; more preferably 8% - 18%.

38. A method for detecting protein phosphorylation in a biological sample, comprising the following steps: obtaining a coacervate composition as described in any of previous claims 1-28; contacting the biological sample with the coacervate composition, preferably for 3 hours at room temperature; imaging the coacervate composition, and / or performing spectroscopic analyses in the coacervate sample containing the biological sample; quantifying the presence of protein phosphorylation.

39. Method according to the previous claim wherein the phosphorylated protein is a Tau protein.

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