Synthetic Supramolecular Systems in Life-like Materials and Protocell Models

Living organisms can be abstracted as complex pools of molecules that work in coordination to create life. Understanding the chemical foundation of life can help explain its evolution and tackle important biochemical challenges at their very roots. Supramolecular chemistry plays a vital role in the structure and function of many cellular components, which chemists are learning to mimic and control with synthetic molecules. Here, we review the design and function of artificial supramolecular systems that imitate cellular structures and responses. The development of such smart materials not only explains the connection between chemistry and biology but also expands our comprehension of life beyond the natural building blocks of cells. Current and future applications of these technologies range from tissue engineering to biomedical nanotechnology and responsive materials. One of the biggest challenges in modern chemistry is the preparation of synthetic materials with life-like behavior for the assembly of artificial cells. In recent years, numerous artificial systems that mimic cellular components and functions have been developed. Supramolecular chemistry plays a key role in such cell mimics given that non-covalent interactions control the shape and function of many biomolecules, such as DNA base pairing, protein structure, ligand-receptor binding, and lipid membrane packing. However, the complexity of living cells constitutes a major challenge for their bottom-up assembly from pure synthetic materials. Inspired by the building blocks of nature, a wide range of supramolecular systems have been developed to reproduce cellular functions such as cell-cell communication, signaling cascades, and dynamic cytoskeleton assemblies. This review surveys a selection of key advances in synthetic derivatives of biomolecules with supramolecular organization and life-like behavior by addressing their non-covalent foundation and integration as increasingly complex protocell models. One of the biggest challenges in modern chemistry is the preparation of synthetic materials with life-like behavior for the assembly of artificial cells. In recent years, numerous artificial systems that mimic cellular components and functions have been developed. Supramolecular chemistry plays a key role in such cell mimics given that non-covalent interactions control the shape and function of many biomolecules, such as DNA base pairing, protein structure, ligand-receptor binding, and lipid membrane packing. However, the complexity of living cells constitutes a major challenge for their bottom-up assembly from pure synthetic materials. Inspired by the building blocks of nature, a wide range of supramolecular systems have been developed to reproduce cellular functions such as cell-cell communication, signaling cascades, and dynamic cytoskeleton assemblies. This review surveys a selection of key advances in synthetic derivatives of biomolecules with supramolecular organization and life-like behavior by addressing their non-covalent foundation and integration as increasingly complex protocell models. The design of simple stimuli-responsive supramolecular systems with life-like behavior constitutes a fundamental scientific exercise toward understanding the role of self-assembly in the origin of life. By “life-like,” we refer to the natural cellular and sub-cellular responses and mechanisms that are fundamental to life from the molecular to macroscopic level (e.g., energy consumption and dissipation, dynamic self-assembly, molecular replication, motion, sensing, and signaling).1Grzybowski B.A. Huck W.T.S. The nanotechnology of life-inspired systems.Nat. Nanotechnol. 2016; 11: 585-592Crossref PubMed Google Scholar Replicating these complex phenomena with simple synthetic building blocks poses a daunting challenge from a supramolecular and functional standpoint. However, minimalistic systems can now be engineered from the bottom-up to imitate and tune particular biological processes.2Göpfrich K. Platzman I. Spatz J.P. Mastering complexity: towards bottom-up construction of multifunctional eukaryotic synthetic cells.Trends Biotechnol. 2018; 36: 938-951Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 3Itel F. Schattling P.S. Zhang Y. Städler B. Enzymes as key features in therapeutic cell mimicry.Adv. Drug Deliv. Rev. 2017; 118: 94-108Crossref PubMed Scopus (23) Google Scholar, 4Bayley H. Building blocks for cells and tissues: beyond a game.Emerg. Top. 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Hyman A. et al.MaxSynBio: avenues towards creating cells from the bottom up.Angew. Chem. Int. Ed. 2018; 57: 13382-13392Crossref PubMed Scopus (50) Google Scholar,6Webber M.J. Appel E.A. Meijer E.W. Langer R. Supramolecular biomaterials.Nat. Mater. 2016; 15: 13-26Crossref PubMed Scopus (617) Google Scholar Supramolecular interactions play a central role in defining the structure and function of biological systems.7Krieg E. Bastings M.M.C. Besenius P. Rybtchinski B. Supramolecular polymers in aqueous media.Chem. Rev. 2016; 116: 2414-2477Crossref PubMed Scopus (289) Google Scholar,8Savyasachi A.J. Kotova O. Shanmugaraju S. Bradberry S.J. Ó’Máille G.M. Gunnlaugsson T. Supramolecular chemistry: a toolkit for soft functional materials and organic particles.Chem. 2017; 3: 764-811Abstract Full Text Full Text PDF Google Scholar The bases of protein folding, DNA hybridization, or membrane integrity rely on the interplay between inter- and intramolecular self-assembly. Therefore, artificial cells made of synthetic building blocks capitalize on mimicking, tuning, and blending these interactions to design artificial suprastructures with life-like organization and environmental responses.5Schwille P. Spatz J. Landfester K. Bodenschatz E. Herminghaus S. Sourjik V. Erb T.J. Bastiaens P. Lipowsky R. Hyman A. et al.MaxSynBio: avenues towards creating cells from the bottom up.Angew. Chem. Int. Ed. 2018; 57: 13382-13392Crossref PubMed Scopus (50) Google Scholar,9Buddingh’ B.C. Hest J.C.M. van. Artificial cells: synthetic compartments with life-like functionality and adaptivity.Acc. Chem. Res. 2017; 50: 769-777Crossref PubMed Scopus (156) Google Scholar Supramolecular forces work cooperatively in living organisms toward stronger interactions with defined spatial arrangement.8Savyasachi A.J. Kotova O. Shanmugaraju S. Bradberry S.J. Ó’Máille G.M. Gunnlaugsson T. Supramolecular chemistry: a toolkit for soft functional materials and organic particles.Chem. 2017; 3: 764-811Abstract Full Text Full Text PDF Google Scholar Importantly, artificial supramolecular systems can be rationally designed to reversibly respond to physical and chemical stimuli, being particularly well suited for mimicking the adaptive behavior of living organisms.10Tu Y. Peng F. Adawy A. Men Y. Abdelmohsen L.K.E.A. Wilson D.A. Mimicking the cell: bio-inspired functions of supramolecular assemblies.Chem. Rev. 2016; 116: 2023-2078Crossref PubMed Scopus (128) Google Scholar Beyond natural biomolecules and derivatives thereof, fully artificial supramolecular designs exploit the principles of non-covalent interactions to open new opportunities in the rational design of life-like synthetic materials and minimal artificial cells. We here survey the latest and most remarkable developments in artificial life-mimicking systems through application of supramolecular chemistry. The following discussion covers the design of artificial building blocks, their supramolecular organization, and their life-like behavior. This review is organized on the basis of the chemical nature of the assembling molecules. First, natural biomolecules (i.e., peptides and proteins, nucleic acids, lipids, and glycans) and derivatives are repurposed for alternative biological functions and/or modulating their natural responses. Second, non-biologically related building blocks (e.g., synthetic polymers, molecular motors, and artificial receptors) will further illustrate the potential of rational supramolecular designs to mimic, tune, and expand the natural responses of cells and tissues. Overall, we aim to provide a broad perspective of the new synthetic suprastructures with life mimicry, as well as address related hot topics in the field, such as out-of-equilibrium self-assembly, supramolecular self-replicators, and kinetic and thermodynamic control.11Mattia E. Otto S. Supramolecular systems chemistry.Nat. Nanotechnol. 2015; 10: 111-119Crossref PubMed Scopus (386) Google Scholar,12Bai Y. Chotera A. Taran O. Liang C. Ashkenasy G. Lynn D.G. Achieving biopolymer synergy in systems chemistry.Chem. Soc. Rev. 2018; 47: 5444-5456Crossref PubMed Google Scholar We have included a selection of key references to refer the reader to more detailed and specific literature on each particular topic. Besides the derived lessons learned from supramolecular systems,13Gasparini G. Bang E.K. Montenegro J. Matile S. Cellular uptake: lessons from supramolecular organic chemistry.Chem. Commun. 2015; 51: 10389-10402Crossref PubMed Google Scholar,14Fuertes A. Juanes M. Granja J.R. Montenegro J. Supramolecular functional assemblies: dynamic membrane transporters and peptide nanotubular composites.Chem. Commun. 2017; 53: 7861-7871Crossref PubMed Google Scholar new biomimicking materials can also bring important applications, such as cell and tissue engineering, diagnostics, and next-generation therapies. The implementation of these technologies in vivo is also presented here to demonstrate their current and future potential. Conceptually, the bottom-up engineering of synthetic cells helps us understand the implications of self-assembly in the origins of life, posing plausible evolutionary steps from simple building blocks to complex and highly specialized biostructures. The diverse chemical functionality and inherent tendency of peptides to assemble (e.g., α helices and β sheets) offer high flexibility to design biomimetic structures.15Wang J. Liu K. Xing R. Yan X. Peptide self-assembly: thermodynamics and kinetics.Chem. Soc. Rev. 2016; 45: 5589-5604Crossref PubMed Google Scholar Inspired by natural protein architectures, artificial peptide scaffolds have been designed to combine hierarchical self-assembly with biomimicry.16Bai Y. Luo Q. Liu J. Protein self-assembly via supramolecular strategies.Chem. Soc. Rev. 2016; 45: 2756-2767Crossref PubMed Google Scholar Importantly, the activity of many natural peptides and proteins is chemically regulated by their metabolic (de)activation, which ultimately translates into conformational changes that dictate function. For example, the eukaryotic cytoskeleton consists of proteins that show alternating cycles of growth and decay triggered by phosphorylation and dephosphorylation events.7Krieg E. Bastings M.M.C. Besenius P. Rybtchinski B. Supramolecular polymers in aqueous media.Chem. Rev. 2016; 116: 2414-2477Crossref PubMed Scopus (289) Google Scholar,17Fletcher D.A. Mullins R.D. Cell mechanics and the cytoskeleton.Nature. 2010; 463: 485-492Crossref PubMed Scopus (1196) Google Scholar This dynamic instability allows the active remodeling of these natural supramolecular fibers with precise spatiotemporal resolution. However, given the structural complexity of cytoskeletal proteins, chemists have focused on the established principles of peptide self-assembly to obtain artificial minimalistic scaffolds that mimic this adaptive behavior. Because of the vast literature in supramolecular bio-inspired peptide systems, we will divide this section into two parts: (1) artificial peptide assemblies fully based on non-covalent interactions and (2) reaction-based systems, where the formation and breaking of covalent bonds control self-assembly. The dynamic fibrillation of short synthetic peptides is one of the most studied prototypes of supramolecular biomimetic assemblies.18Sato K. Hendricks M.P. Palmer L.C. Stupp S.I. Peptide supramolecular materials for therapeutics.Chem. Soc. Rev. 2018; 47: 7539-7551Crossref PubMed Google Scholar The rationally predictable non-covalent interactions of short peptides give chemists the opportunity to design supramolecular structures with complex life-like behavior, such as dynamic remodeling and self-replication. One of the benchmark artificial fibrillating peptides are Stupp’s peptide amphiphiles (PAs), which consist of aliphatic chains conjugated to the termini of short peptides with hydrophobic β sheet inductors and charged residues (Figure 1A).19Hendricks M.P. Sato K. Palmer L.C. Stupp S.I. Supramolecular assembly of peptide amphiphiles.Acc. Chem. Res. 2017; 50: 2440-2448Crossref PubMed Scopus (119) Google Scholar In these PAs, the aliphatic tail is the main driving force of self-assembly by hydrophobic effect in water. Additionally, the peptide region forms stabilizing hydrogen (H)-bonded networks, whereas charged residues maintain the extended conformation of the assembly by electrostatic repulsion. A recent expansion of this work exploits the conjugation of fibrillating PAs to DNA, which generates intertwined PA fibers by complementary DNA pairing (Figures 1A and 1B).20Freeman R. Han M. Álvarez Z. Lewis J.A. Wester J.R. Stephanopoulos N. McClendon M.T. Lynsky C. Godbe J.M. Sangji H. et al.Reversible self-assembly of superstructured networks.Science. 2018; 362: 808-813Crossref PubMed Scopus (49) Google Scholar These PA-DNA hybrids self-assemble into dynamic 3D structures that allow reversible remodeling, and these function as synthetic mimics of the extracellular matrix. Hence, specific DNA hybridization endows self-assembling peptides with additional supramolecular recognition and hierarchical organization, imitating the different levels of non-covalent organization found in biological systems. Supramolecular recognition is proposed as one of the mechanisms that might have guided the chemical evolution of life given that certain molecules might have been selected and replicated through self-assembly from pools of structural analogs.11Mattia E. Otto S. Supramolecular systems chemistry.Nat. Nanotechnol. 2015; 10: 111-119Crossref PubMed Scopus (386) Google Scholar,23Ruiz-Mirazo K. Briones C. Escosura A. de la. Chemical roots of biological evolution: the origins of life as a process of development of autonomous functional systems.Open Biol. 2017; 7: 170050Crossref PubMed Scopus (20) Google Scholar Otto and co-workers have developed intriguing peptide-based self-replicating dynamic libraries, which arise from short synthetic peptides connected to an aromatic di-thiol that oligomerizes into macrocyclic products (Figure 1C).21Sadownik J.W. Mattia E. Nowak P. Otto S. Diversification of self-replicating molecules.Nat. Chem. 2016; 8: 264-269Crossref PubMed Scopus (98) Google Scholar The kinetic products obtained from these peptides are short macrocycles (3-mer and 4-mer), which over time rearrange into larger ones (e.g., 6-mer) that self-assemble into fibers driven by β sheet interactions. The supramolecular stacking of 6-mers templates shorter macrocycles and drives their evolution to the self-assembling product. Interestingly, the self-replication of the 6-mer only takes place when the system is agitated, as shear stress causes pre-formed fibers to fragment into nucleation points for the selection and elongation of 6-mer fibers.24Colomb-Delsuc M. Mattia E. Sadownik J.W. Otto S. Exponential self-replication enabled through a fibre elongation/breakage mechanism.Nat. Commun. 2015; 6: 7427Crossref PubMed Scopus (47) Google Scholar These dynamic combinatorial libraries allow the study of not only molecular Darwinian evolution, as just described, but also other complex inter-species relationships, such as parasitic and predatory behavior with the same chemical foundation.25Altay M. Altay Y. Otto S. Parasitic behavior of self-replicating molecules.Angew. Chem. Int. Ed. 2018; 57: 10564-10568Crossref PubMed Scopus (12) Google Scholar Alternatively to disulfide chemistry, Lynn and co-workers developed analogous dynamic peptide networks by using reversible N,N-acetal linkages for oligomer exchange (Figure 1D).22Chen C. Tan J. Hsieh M.-C. Pan T. Goodwin J.T. Mehta A.K. Grover M.A. Lynn D.G. Design of multi-phase dynamic chemical networks.Nat. Chem. 2017; 9: 799-804Crossref PubMed Google Scholar Over time, the ability of the linear trimers to fibrillate as β sheets drives their self-replication and supramolecular selection from a pool of oligomers in equilibrium. Template-directed growth was confirmed by the rapid fibrillation of the monomer when seeded with a pre-assembled trimer. Remarkably, this dynamic system could also grow supramolecular nanotubes from seeds of a related amyloid peptide, demonstrating the flexibility of this network to replicate different templates. The group also demonstrated the on-surface catalytic activity of these supramolecular phases, which despite being unable to read or write specific sequences can perform templated polymerizations that resemble the function of natural polymerases.26Omosun T.O. Hsieh M.-C. Childers W.S. Das D. Mehta A.K. Anthony N.R. Pan T. Grover M.A. Berland K.M. Lynn D.G. Catalytic diversity in self-propagating peptide assemblies.Nat. Chem. 2017; 9: 805-809Crossref PubMed Google Scholar One step beyond one-dimensional (1D) peptide fibrillation was taken with the design of de novo coiled-coil peptide assemblies, which opened up access to more complex hierarchical structures and life-like functions. Woolfson and co-workers have contributed extensively to this area with de novo coiled-coil peptide barrels that work as synthetic receptors for small hydrophobic molecules27Thomas F. Dawson W.M. Lang E.J.M. Burton A.J. Bartlett G.J. Rhys G.G. Mulholland A.J. Woolfson D.N. De novo-designed α-helical barrels as receptors for small molecules.ACS Synth. Biol. 2018; 7: 1808-1816Crossref PubMed Scopus (14) Google Scholar and display artificial esterase activity28Burton A.J. Thomson A.R. Dawson W.M. Brady R.L. Woolfson D.N. Installing hydrolytic activity into a completely de novo protein framework.Nat. Chem. 2016; 8: 837-844Crossref PubMed Google Scholar (Figures 2A and 2B ). These multimeric coiled coils consist of amphiphilic α-helical peptides that follow the heptad design “hpphppp,” where “h” and “p” correspond to hydrophobic and polar amino acids, respectively, thus creating a hydrophobic face in the helix that packs into a pore upon oligomerization. Furthermore, de novo coiled-coil peptides could direct the self-assembly of natural proteins into artificial supramolecular scaffolds within living bacteria, where tagged enzymes could be accumulated with improved turnover rates.29Lee M.J. Mantell J. Hodgson L. Alibhai D. Fletcher J.M. Brown I.R. Frank S. Xue W.-F. Verkade P. Woolfson D.N. Warren M.J. Engineered synthetic scaffolds for organizing proteins within the bacterial cytoplasm.Nat. Chem. Biol. 2018; 14: 142-147Crossref PubMed Scopus (43) Google Scholar Jerala and co-workers have encoded multiple coiled-coil interactions into long peptides—over 700 amino acids—that fold into defined polyhedra, such as tetrahedra, pyramids, and prisms (Figure 2C).30Ljubetič A. Lapenta F. Gradišar H. Drobnak I. Aupič J. Strmšek Ž. Lainšček D. Hafner-Bratkovič I. Majerle A. Krivec N. et al.Design of coiled-coil protein-origami cages that self-assemble in vitro and in vivo.Nat. Biotechnol. 2017; 35: 1094-1101Crossref PubMed Scopus (0) Google Scholar Beyond the fundamental expansion of non-natural supramolecular peptide assemblies, the authors demonstrate the expression and correct folding of these coiled-coil peptide origami from plasmids in vivo and hence their potential application in biological settings and in situ generation. This group has also implemented de novo coiled-coil peptides as substrates for proteolytic logic circuits, which are versatile tools for emulating cellular signaling pathways and thus studying the dynamics and evolution of biological regulation.31Fink T. Lonzarić J. Praznik A. Plaper T. Merljak E. Leben K. Jerala N. Lebar T. Strmšek Ž. Lapenta F. et al.Design of fast proteolysis-based signaling and logic circuits in mammalian cells.Nat. Chem. Biol. 2018; 15: 115-122Crossref PubMed Scopus (23) Google Scholar Engineering supramolecular protein interactions de novo in coiled-coil systems has also allowed the expression and orthogonal assembly of coiled-coil heterodimers in living bacteria.32Chen Z. Boyken S.E. Jia M. Busch F. Flores-Solis D. Bick M.J. Lu P. VanAernum Z.L. Sahasrabuddhe A. Langan R.A. et al.Programmable design of orthogonal protein heterodimers.Nature. 2019; 565: 106-111Crossref PubMed Scopus (29) Google Scholar Unlike most hydrophobic-driven protein oligomers, these coiled-coil heterodimers were established by highly specific H-bonding patterns between side chains reminiscent of natural DNA base pairing. Moreover, Ghadiri and co-workers first demonstrated the autonomous self-replication of coiled-coil peptide dimers via supramolecular templation of activated precursor fragments through leucine zippers.33Lee D.H. Granja J.R. Martinez J.A. Severin K. Ghadiri M.R. A self-replicating peptide.Nature. 1996; 382: 525-528Crossref PubMed Scopus (0) Google Scholar This concept was recently applied to a reversible self-replicating coiled-coil network, where dynamic thioester bonds between peptide fragments allowed the transition between coiled-coil and unfolded precursor states.34Maity I. Wagner N. Mukherjee R. Dev D. Peacock-Lopez E. Cohen-Luria R. Ashkenasy G. A chemically fueled non-enzymatic bistable network.Nat. Commun. 2019; 10: 4636Crossref PubMed Scopus (5) Google Scholar The alternative use of d-amino acids as peptide building blocks opens up access to interesting non-natural supramolecular architectures, such as one-faced β sheets35Garcia A.M. Iglesias D. Parisi E. Styan K.E. Waddington L.J. Deganutti C. De Zorzi R.D. Grassi M. Melchionna M. Vargiu A.V. Marchesan S. Chirality effects on peptide self-assembly unraveled from molecules to materials.Chem. 2018; 4: 1862-1876Abstract Full Text Full Text PDF Scopus (41) Google Scholar and cyclic peptide nanotubes.36Montenegro J. Ghadiri M.R. Granja J.R. Ion channel models based on self-assembling cyclic peptide nanotubes.Acc. Chem. Res. 2013; 46: 2955-2965Crossref PubMed Scopus (171) Google Scholar Our group has worked extensively on d/l-alternating cyclic peptides that stack by H bonding into nanotubes, allowing additional supramolecular interactions encoded on their superficial side chains to control self-assembly.14Fuertes A. Juanes M. Granja J.R. Montenegro J. Supramolecular functional assemblies: dynamic membrane transporters and peptide nanotubular composites.Chem. Commun. 2017; 53: 7861-7871Crossref PubMed Google Scholar Thus, the fibrillation of cyclic peptides can be controlled with pH, when charge repulsions between protonated histidine residues are neutralized by the addition of base (Figure 3A).37Méndez-Ardoy A. Granja J.R. Montenegro J. pH-triggered self-assembly and hydrogelation of cyclic peptide nanotubes confined in water micro-droplets.Nanoscale Horiz. 2018; 3: 391-396Crossref PubMed Google Scholar In this example, lateral hydrophobic and π-π interactions between pendant pyrene units drove the bundling of peptide nanotubes into larger fibers. Alternatively, charge repulsions between cyclic peptides can be screened by the addition of small electrolytes to induce nanotube formation and bundling.38Méndez-Ardoy A. Bayón-Fernández A. Yu Z. Abell C. Granja J.R. Montenegro J. Spatially controlled supramolecular polymerization of peptide nanotubes by microfluidics.Angew. Chem. Int. Ed. 2020; 59: 6902-6908Crossref PubMed Scopus (0) Google Scholar The confined and localized fibrillation of these cyclic peptides at the center or interface of droplets emulates the spatially controlled supramolecular polymerization of a cytoskeleton (Figure 3B). Furthermore, segregation of polar and hydrophobic amino acids in cyclic peptides allows the amplification of this amphiphilic character in one dimension along the resulting nanotubes (Figure 3C).39Insua I. Montenegro J. 1D to 2D self assembly of cyclic peptides.J. Am. Chem. Soc. 2020; 142: 300-307Crossref PubMed Scopus (4) Google Scholar These amphiphilic nanotubes can further self-assemble as bilayers in aqueous medium by hydrophobic packing, where leucine zippers provide directional growth in this second dimension. Hierarchical 1D and 2D self-assembly can be thus engineered into d/l-alternating cyclic peptides to form giant supramolecular nanosheets with stimuli-responsive behavior at a high-micron scale. These novel 2D materials provide alternative supramolecular non-lipid membranes, also accessible from other peptide-based building blocks, such as PAs,40Lin Y. Thomas M.R. Gelmi A. Leonardo V. Pashuck E.T. Maynard S.A. Wang Y. Stevens M.M. Self-assembled 2D free-standing Janus nanosheets with single-layer thickness.J. Am. Chem. Soc. 2017; 139: 13592-13595Crossref PubMed Scopus (0) Google Scholar triple helices,41Merg A.D. Touponse G. Genderen E. van Zuo X. Bazrafshan A. Blum T. Hughes S. Salaita K. Abrahams J.P. Conticello V.P. 2D crystal engineering of nanosheets assembled from helical peptide building blocks.Angew. Chem. Int. Ed. 2019; 58: 13507-13512Crossref PubMed Scopus (3) Google Scholar and peptoids.42Battigelli A. Kim J.H. Dehigaspitiya D.C. Proulx C. Robertson E.J. Murray D.J. Rad B. Kirshenbaum K. Zuckermann R.N. Glycosylated peptoid nanosheets as a multivalent scaffold for protein recognition.ACS Nano. 2018; 12: 2455-2465Crossref PubMed Scopus (19) Google Scholar The fibrillation of short synthetic peptides driven by enzymatic transformations represents a minimal model to study the regulated dynamics of biological suprastructures. For example, intracellular fibrillation of a synthetic peptide was achieved in vitro by sequential dephosphorylation by alkaline phosphatase (ALP) and disulfide reduction of the peptide precursor, modifications that removed Coulombic repulsions and thus triggered intracellular self-assembly (Figure 4A).43Zhan J. Cai Y. He S. Wang L. Yang Z. Tandem molecular self-assembly in liver cancer cells.Angew. Chem. Int. Ed. 2018; 57: 1813-1816Crossref PubMed Scopus (39) Google Scholar Similarly, cellular production of ALP can also trigger the extracellular fibrillation of short aromatic peptides, which undergo π-π and hydrophobic packing upon removal of sacrificial phosphate groups.44Zhou J. Du X. Yamagata N. Xu B. Enzyme-instructed self-assembly of small D-peptides as a multiple-step process for selectively killing cancer cells.J. Am. Chem. Soc. 2016; 138: 3813-3823Crossref PubMed Scopus (117) Google Scholar These are excellent examples of how rational design and self-assembly can exploit cellular metabolism to engineer artificial supramolecules in living cells. Enzymes catalyzing both amide formation and hydrolysis can be employed to generate transient peptide fibers regulated by the kinetics of these two enzymatic steps. Ulijn and co-workers illustrated this concept with the conversion of soluble aspartame into fibrillating tripeptides by action of α-chymotrypsin, which also catalyzed the hydrolysis of the self-assembling tripeptide to give an inactive product (Figure 4B).45Pappas C.G. Sasselli I.R. Ulijn R.V. Biocatalytic pathway selection in transient tripeptide nanostructures.Angew. Chem. Int. Ed. 2015; 54: 8119-8123Crossref PubMed Scopus (0) Google Scholar This group later employed α-chymotrypsin to control the self-assembly of a chimeric dipeptide, either as transient helical fibers or as nanotubes, by feeding amino acids of specific polarity and chirality that determined enzymatic hydrolysis and amidation rates.47Kumar M. Ing N.L. Narang V. Wijerathne N.K. Hochbaum A.I. Ulijn R.V. Amino-acid-encoded biocatalytic self-assembly enables the formation of transient conducting nanostructures.Nat. Chem. 2018; 10: 696-703Crossref PubMed Scopus (53) Google Scholar Thus, the competition between the supramolecular or covalent (i.e., enzymatic) attachment of the fed amino acids to the parent dipeptide selected for specific supramolecular pathways with kinetic control according to α-chymotrypsin’s reactivity. Importantly, the sustained activity of this enzyme accumulates reaction byproducts that lead to a progressive drop in turnover rates. The inhibition of enzymatic pathways by their own byproducts is a problem solved in living organisms with waste-clearing mechanisms. To mimic the natural perpetuation of fueled supramolecular systems, Hermans and co-workers developed a peptide-based supramolecular dissipative system where enzymatic (de)phosphorylation of a perylenediimide (PDI) peptide derivative induces the formation of transient fibers inside a dialysis chamber, which can be sustained over time by a constant supply of fuel (i.e., ATP) and waste removal.47Kumar M. Ing N.L. Narang V. Wijerathne N.K. Hochbaum A.I. Ulijn R.V. Amino-acid-encoded biocatalytic self-assembly enables the formation of transi