Nature Chemistry

On-demand assembly of arbitrary, sequence-defined polymers from a pool of monomers is a major challenge in modern chemistry, towards which limited progress has been made. By contrast, biological systems routinely use information-bearing DNA and RNA templates to catalytically synthesize a precise, far-from-equilibrium ensemble of nucleic acid and protein sequences from the available pools of NTPs or aminoacyl-tRNAs. Inspired by these biological examples, we introduce an enzyme-free DNA strand displacement network in which single-stranded DNA sequences template the assembly of specific non-covalent DNA multimers of up to length five, under isothermal and autonomous conditions. The templates demonstrate significant turnover, bypassing product inhibition. They can thereby catalyse the formation of a far-from-equilibrium ensemble of long-lived metastable products that are not otherwise addressable.

Adenosine 5-triphosphate (ATP) is found to form biomolecular condensates with proteins. However, without complementary proteins, the small size and high charge density of ATP molecules create substantial electrostatic and entropic barriers that prevent them from forming condensates. Here, we find that macromolecular crowding overcomes these energetic barriers, promoting ATP molecules to self-associate and form protein-free liquid-like condensates through screened electrostatic repulsion and enhanced hydrogen bonding. Importantly, ATP condensates are responsive to multiple stimuli and create distinct microenvironments that selectively enrich various guest molecules and protect ribonucleic acids from DNAzyme cleavage. These findings uncover important roles of ATP in forming dynamic, chemically distinct condensates via homotypic interactions, potentially expanding its classical view beyond a canonical energy carrier to a structural and regulatory architect in cellular physiology and prebiotic chemistry.

Dynamic biomolecular condensates play crucial roles in intracellular compartmentalization and physiological functions. While engineering tools for compartmentalization have expanded add-on functionalities, directly amplifying the inherent catalytic machinery within biological phase-separated droplets has remained elusive. Herein, we developed a phase-separated oxidative folding reaction chamber based on protein disulfide isomerase A6 (PDIA6) by chemically targeting its active site CxxC motif to enhance enzymatic activity within PDIA6 droplets. A para-substituted N-methylated pyridinylmethanethiol (pMePySH) enhanced the catalytic oxidative folding of bovine pancreatic trypsin inhibitor, proinsulin, and antibody up to 12-fold within in vitro PDIA6 droplets. Furthermore, pMePySH targeted PDIA6 foci within the endoplasmic reticulum, significantly promoting insulin secretion. These findings offer a powerful platform for the spatiotemporal manipulation of protein folding, with profound implications for the scalable manufacturing of therapeutic antibodies and other complex biopharmaceuticals.

Biomolecular condensates are functionally gated by their material properties. While RNA is a primary structural scaffold, its sequence-specific interactions can aberrantly drive condensates into dysfunctional solid aggregates. Yet, actively reversing this solidification to restore function remains a fundamental challenge, impeding progress in synthetic biology and therapeutics interventions. Here, we establish that complex coacervates can be engineered as liquefiers to actively remodel solid RNA-peptide aggregates into functional liquid droplets. Integrating systematic experiments with all-atom simulations, we decode a multiscale mechanism: coacervate infiltration at the micrometer scale mediates molecular buffering at the nanometer scale, which fluidizes the crosslinked network to drive macroscopic liquefaction. This capability is governed by a design rule, where coacervates formed by small-molecular anions exhibit optimal efficacy. We deploy this principle to functionally rescue silenced RNA within a model of pathologically solidified ribonucleoprotein assemblies. Our work provides a general framework for the active, compositional control of biomolecular phase behavior, with direct implications for managing pathological aggregation and engineering functional condensates in synthetic and living systems.

We report the discovery that chemical reactions can be catalyzed by condensates formed by intrinsically disordered proteins (IDPs). The proteins themselves lack any catalytic activities. Catalytic functions of condensates emerge as a consequence of sequence-dependent mesoscale electrochemical microenvironments created by phase separation. Stimulated Raman spectroscopy suggests that the catalytic behaviors of condensates are attributable to the spatial variations of water activities across condensate interiors and interfaces. We show that condensates are capable of catalyzing diverse cellularly relevant hydrolysis reactions. Through sequence design, the electrochemical properties of condensates can be programmed to exert control over catalytic behaviors. Incorporation of synthetic condensates into live cells alters transcription profiles and enables the activation of gene circuits that depend on products of hydrolysis reactions catalyzed by condensates. Our discovery of suggests that condensates, depending on their composition-dependent electrochemical properties, can be "Condenzymes", which contribute unexpected emergent chemical functions in cells.

Direct solar-to-chemical conversion offers a compelling route to clean, dispatchable energy. Photosystem I (PSI), an evolutionarily optimized light-driven oxidoreductase central to oxygenic photosynthesis, can be repurposed for direct solar-fuel production by efficiently coupling its photochemistry to catalysts, thereby storing sunlight as chemical energy in the H-H bond of H2. One promising architecture integrates PSI with Pt nanoparticle (PtNP) catalysts to create photocatalytic PSI-PtNP biohybrids. Advancing these systems requires molecular-level insight into protein-nanoparticle interactions and the bio-nano electron transfer pathways that govern activity; however, progress has been constrained by limited structural data to guide rational design. Here, we present two molecular structures of active PSI-PtNP assemblies that (a) compare thermophilic and mesophilic PSI scaffolds and (b) probe how removal of the terminal [4Fe-4S] clusters and stromal subunits in PSI reshapes protein-nanoparticle interfaces and photocatalysis. Structural analyses and molecular dynamics simulations define the interface topology, electrostatics, and cofactor-to-nanoparticle distances, revealing key molecular features that control biohybrid formation and electron transfer efficiency. These data establish mechanistic links between scaffold composition, bio-nano interface geometry, and catalytic performance, yielding design principles for optimizing PSI-PtNP architectures. The resulting structure-function insights provide a blueprint for engineering PSI-based solar-fuels systems and, more broadly, inform the design of protein-nanomaterial interfaces for light-driven catalysis.

Polyethylene terephthalate (PET) is a widely used thermoplastic whose high crystallinity poses a major barrier to upscaling enzymatic recycling. While PETases with high activity and stability have been reported, no enzyme capable of directly depolymerizing crystalline PET (cPET) has been discovered, and the molecular determinants limiting their efficacy remain difficult to characterize. Here, we integrate experimental conformational ratios of crystalline and amorphous PET chains with enhanced-sampling molecular dynamics simulations to map the free-energy landscape of a prototypical PETase bound to PET oligomers, revealing how structural equilibria translate to catalytic function. Surprisingly, productive enzyme-substrate catalytic configurations can be reached for both crystalline and amorphous PET chains. However, forming catalytic ensembles with cPET requires ~25 kJ/mol more than with aPET, with an additional ~17 kJ/mol per monomer needed for chain separation, which further limits enzymatic activity on crystalline substrates. The model highlights limitations of current alpha/beta-hydrolase scaffolds used for PET depolymerization and indicates directions for their redesign to enable cPET depolymerization. Our approach showcases a general strategy to explore substrate-enzyme catalytic ensembles in plastic depolymerization and guide enzyme design with built-in sustainability.

Self-assembling single-chain amphiphiles available in the prebiotic environment likely played a fundamental role in the advent of primitive cell cycles. However, the instability of prebiotic fatty acid-based membranes to temperature and pH seems to suggest that primitive cells could only host prebiotically-relevant processes in a narrow range of non-fluctuating environmental conditions. Here we propose a novel primitive cell cycle driven by environmental fluctuations, which enable the generation of daughter protocells with reshuffled content. A reversible membrane-to-oil phase transition accounts for the dissolution of fatty acid-based vesicles at high temperatures, and the concomitant release of genetic content. At low temperatures, fatty acid bilayers reassemble and encapsulate reshuffled genetic material in a new cohort of protocells. Notably, we find that our disassembly/reassembly cycle drives the emergence of functional RNA-containing primitive cells from parent non-functional compartments. Thus, by exploiting the intrinsic instability of prebiotic fatty acid vesicles, our results point at an environmentally-driven tunable primitive cell cycle, which supports the release and reshuffle of protocellular genetic and membrane components, potentially leading to a new generation of protocells with superior traits. In the absence of protocellular transport machinery, the environmentally-driven disassembly/assembly cycle proposed herein would have supported genetic content reshuffling transmitted to primitive cell progeny, hinting at a potential mechanism important to initiate Darwinian evolution of early lifeforms.

Phosphorylation is a major regulator of biomolecular condensation, yet it remains unclear whether clustered phosphoserines can directly tune phase behavior via metal-ion coordination. Here, using solution NMR spectroscopy and human heterochromatin protein 1 (HP1) as a model system, we show that stepwise phosphorylation of its N-terminal serine cluster generates a dynamic metal-responsive module that engages Mg{superscript 2}, Ca{superscript 2}, and Mn{superscript 2}, whereas the unmodified protein shows little or no response. Metal coordination lowers the saturation concentration of phosphorylated HP1, reshapes the temperature-dependent stability of its condensates, and modulates the effects of peptide regulators in an ion-specific manner. Our data support a model in which weak, transient metal-mediated contacts enhance intermolecular connectivity between phosphorylated HP1 molecules, promoting reversible condensation alongside canonical electrostatic interactions. These findings establish clustered phosphoserines as sequence-encoded metal-responsive elements that couple post-translational modification to the material properties of biomolecular condensates.

The emergence of respiratory, photosynthetic, and assimilatory complexes in evolution required proteins capable of binding multiple catalytic and electron-transfer cofactors while exerting fine control over their spatial arrangement. Across natural systems these cofactors are preferentially positioned in loop regions. In contrast, most protein design strategies have focused on installing cofactor-binding sites within helical elements. Here we show that introducing only a pair of appropriately placed histidine ligands into the interhelical loop regions of a canonical single-chain four-helix bundle is sufficient to create new well-defined high affinity heterocofactor binding sites. This simple modification enables the self-assembly of complexes containing up to three distinct cofactors in a single designed domain with positional specificity. Using this strategy, we creat-ed constructs containing one or two hemes in combination with Zn(II) phthalocyanine monosulfonate, Zn-heme, and the light-harvesting Zn(II) tetraphenylporphyrin tetrasulfonate. Fluorescence measurements of constructs containing the latter show efficient energy transfer between photoactive donor cofactors. By demonstrating that loop-embedded ligands support robust, modular, and evolutionarily plausible cofactor recruitment, this work provides a mechanistic explanation for the widespread placement of redox and catalytic cofactors in loops in natural proteins: only limited packing complementarity is needed, meaning that just a few mutations can introduce a functional cofactor binding site, after which additional mutations can tune affinity, reactivity, and specificity. More importantly, it establishes a straightforward path toward constructing func-tional protein domains that mirror the complexity of biological energy-conversion architectures.

Insulin glargine is formulated at acidic pH but acts after transferring to near-neutral tissue, where its prolonged effect is commonly attributed to isoelectric depot formation. However, the structural pathway linking precipitation to delayed release has remained unresolved. Here we combine ambient-temperature serial femtosecond crystallography, solution biophysics, and multiscale network analyses to define the pH-dependent conformational landscape of hexameric glargine across pH 8.4, 7.3, 6.4, and 5.1. We resolve full hexameric glargine structures and identify a previously unreported, pH-coupled lattice transition from P1211 (near-neutral) to R3:H (acidic), accompanied by redistribution from compact phenolic Rf6-state assemblies to more plastic yet structurally coherent TRf/T3Rf3 states. This transition is accompanied by B-chain N-terminal unpeeling, phenol-pocket collapse, hydration loss, and electrostatic rewiring, and is mirrored in solution by oligomeric heterogeneity, Raman amide-I broadening, reduced thermal stability, and a blue-shifted intrinsic fluorescence maximum. Multiscale analyses further indicate that acidification does not create a new dynamical regime but reweighs pre-existing collective modes along a continuous free-energy landscape. These results support a revised mechanism in which isoelectric precipitation and delayed dissociation are mechanistically coupled through structurally organized molten-like intermediate states, linking glargine pharmacology to intrinsic allosteric redistribution within the hexamer. These findings establish a structural blueprint for benchmarking biosimilar glargine and for engineering next-generation basal insulins by tuning allosteric plasticity and intermediate-state stability.

DNA and RNA are compartmentalized into distinct, heterogeneous structures within cells. However, the separation between RNA and DNA, especially in the absence of modern transcription machinery, is inherently hindered by thermodynamic constraints such as complementary base pairing and entropic mixing. Here, we demonstrate that a single atomic difference in the pentose sugars of single-stranded DNA and RNA with identical sequences profoundly alters their interactions with cationic peptides, driving DNA-RNA segregation to form multiphase condensates. We reveal that the interplay between homotypic and heterotypic interactions among DNA, RNA and peptides dictates the formation of multiphase condensates. Leveraging this mechanism, we design a library of sequence-specific oligonucleotides, termed SEGREGamers, that enable programmable multiphase droplets with coexisting DNA-rich and RNA-rich domains. These synthetic condensates recapitulate key features of cellular nuclear compartments, including selective partitioning of small molecules, promotion of RNA aptamer folding, and enhancement of catalytic RNA cleavage. Our findings highlight the pivotal role of pentose sugar variations in nucleic acid compartmentalization, offering new insights into the evolutionary origins of nuclear organization and providing a versatile platform for engineering synthetic nuclear mimics, programmable molecular condensates, and RNA delivery vehicles.

Messenger RNA (mRNA)-based therapeutics have emerged as a new class of biological medicines, clearly exemplified by the global deployment of mRNA vaccines against the COVID-19 pandemic. Currently, therapeutic mRNA is primarily produced through in vitro transcription that suffers high production costs. Until now, intracellular manufacture of mRNA has been challenging due to the presence of ubiquitous RNases in vivo. Here, we have developed a new approach that protects eukaryotic mRNA from RNase degradation ensuring longevity and integrity of mRNA inside microbial cells. Through targeted strain and molecular engineering, our approach involves specially designed inserts in mRNA that facilitate formation of stabilized and protected protein-mRNA complexes. In addition to vastly improved stability, the protein-mRNA complexes enable convenient purification of mRNA from cell lysate with high purity using conventional chromatography. The work reported here promises a scalable, rapid, and low-cost approach to produce fully functional eukaryotic mRNA using well-known microbial systems.

Lasso peptides adopt a distinctive [1]rotaxane conformation, yet the principles governing the folding of this kinetically trapped structure have remained elusive. Here, we integrated extensive molecular dynamics simulations and deep learning to elucidate the de novo folding mechanism of 20 lasso peptides lacking secondary post-translational modifications. We constructed Multi-Ensemble Markov Models for each lasso peptide and uncovered a universal uphill folding landscape with spontaneous folding probabilities consistently below 0.8%. Loop stability strongly correlated with folding propensity, and targeted experiments further validated that enhancing loop {beta}-hairpin formation promotes folding of microcin J25, the well-studied lasso peptide extensively characterized as an in vitro model. Additionally, the substantial entropy cost opposed lasso peptide folding. Simulations mimicking enzymatic spatial confinement reduced this penalty and stabilize folding. Leveraging Variational AutoEncoder-based pathway clustering, we resolved distinct pathway channels and representative folding pathways. Together, these findings establish representative folding models and fundamental thermodynamic and kinetic principles for rational engineering of lasso peptides.

Biomolecular condensates have emerged as prominent regulators of dynamic subcellular organisation and essential biological processes. Temperature, in particular, exerts a significant influence on the formation and behaviour of biomolecular condensation. For example, during cellular heat stress, stress granules (SGs) are formed from RNA-binding proteins (RBPs) and RNA, forming liquid condensates to protect the RNA from damage. However, the molecular mechanisms leading to changes in protein phase behaviour are not well understood. To answer how temperature modulates protein interactions and phase behaviour, we developed a high-throughput microfluidic platform, capable of mapping the phase space and quantifying protein interactions in a temperature-dependent manner. Specifically, our approach measures high-resolution protein phase diagrams as a function of temperature, while accurately quantifying changes in the binodal, condensate stoichiometry and free energy contribution of a solute, hence, providing information about the underlying mechanistic driving forces. We employ this approach to investigate the effect of temperature changes on the phase separation of the stress granule scaffold protein Ras GTPase-activating protein-binding protein 1 (G3BP1) with PolyA-RNA. Surprisingly, we find that the G3BP1/RNA phase boundary remains unaffected by the increasing temperature but the underlying stoichiometry and energetics shift, which can only be revealed with high-resolution phase diagrams. This indicates that temperature-induced dissolution is counteracted by entropic processes driving phase separation. With increasing temperature, the G3BP1 content in condensates decreases alongside with a reduction of the free energy of protein interactions, while the RNA content increases driven by entropically favoured hydrophobic interactions. In the context of cellular heat SG formation, these findings could indicate that during heat shock, elevated temperatures directly induce RNA recruitment to stress granules as a cytoprotective mechanism by finetuning the strength of protein and RNA interactions.

The capacity of biomolecular condensates to establish and modulate electrochemical equilibria is emerging as an important functioning mechanism in cellular biochemistry. However, the physical chemistry basis of the electric potentials arising from biomacromolecular phase transitions remains unclear. Here, we show that asymmetry in hydrophobicity, which is a generalizable feature in condensate system, can directly encode an electric potential gradient between the dilute and the dense phases. We demonstrate that using a non-charged intrinsically disordered protein, ion-dependent kosmotropic effect can encode measurable pH and interphase potential gradients into condensate. All-atom molecular dynamics simulations further reveal that the distinct intrinsic transfer free energy of ions defines the ion partitioning capability of condensates via favorable interactions with protein backbones. The simulation also shows the existence of both interfacial and interphase electric potentials. These built-in potentials modulate the partitioning and reactivity of charged solutes, enabling non-enzymatic, potential-dependent chemistry within condensates. Our findings identify hydrophobic asymmetry as a simple and generalizable mechanism for charging biological matter, linking water activity and ion energetics to the emergent electrochemistry of condensates.

Biomolecular condensation via liquid-liquid phase separation of intrinsically disordered proteins/regions (IDPs/IDRs) along with other biomolecules is thought to govern critical cellular functions, whereas, aberrant phase transitions are associated with a range of deadly neurodegenerative diseases. Here we show, a naturally occurring pathological truncation variant of the prion protein (PrP) by a mutation of a tyrosine residue at 145 to a stop codon (Y145Stop) yielding a highly disordered N-terminal IDR that spontaneously phase-separates into liquid-like droplets. Phase separation of this N-terminal segment that is rich in positively charged and aromatic residues is promoted by the electrostatic screening and a multitude of other transient, intermolecular, noncovalent interactions. Single-droplet Raman measurements in conjunction with an array of bioinformatic, spectroscopic, microscopic, and mutagenesis studies revealed that the intrinsic disorder and dynamics are retained in the liquid-like condensates. Lower concentrations of RNA promote the phase transition of Y145Stop at low micromolar protein concentrations under physiological condition. Whereas, higher RNA to protein ratios inhibit condensation indicating the role of RNA in modulating the phase behavior of Y145Stop. Highly dynamic liquid-like droplets eventually transform into dynamically-arrested, ordered, {beta}-rich, amyloid-like aggregates via liquid-to-solid transition upon aging. These amyloid-like aggregates formed via phase separation display the self-templating characteristic and are capable of recruiting and autocatalytically converting monomeric Y145Stop into amyloid fibrils. In contrast to this disease-associated intrinsically disordered Y145 truncated variant, the wild-type full-length PrP exhibited a much lower propensity for phase separation and liquid-to-solid maturation into amyloid-like aggregates hinting at a potentially crucial, chaperone-like, protecting role of the globular C-terminal domain that remains largely conserved in vertebrate evolution. Such an intriguing interplay in the modulation of the protein phase behavior will have much broader implications in cell physiology and disease.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins underlies the formation of biomolecular condensates that regulate diverse cellular processes, while its dysregulation contributes to protein aggregation and disease. Despite its importance, molecularly defined and target-specific strategies to control LLPS remain limited. Here, we present a systematic framework for designing de novo peptides that induce and modulate LLPS of -synuclein. By integrating deep mutational scanning with peptide screening, we identified sequence features that govern condensate formation and enabled the creation of optimized peptides with high efficiency and specificity. Biophysical analyses revealed that LLPS efficiency is dictated by the interplay of solubility, multivalency, and cooperative interactions, resulting in a distinctive bell-shaped phase diagram. Thermodynamic measurements and imaging-based analyses further demonstrated that condensate stability and material properties can be rationally tuned through peptide optimization. Together, these findings establish generalizable design principles for engineering LLPS modulators in biologically and pathologically relevant protein systems.

Catalytic molecular templating, wherein a copolymer molecule serves as a sequence-specific template to propagate genetic information to a daughter copolymer, is fundamental to cells. Templating underlies DNA replication, RNA transcription and protein translation, underpinning the molecular basis of heredity, evolution, and biological function, and allowing staggering complexity to arise from simple building blocks. It has hitherto been challenging to emulate templating without highly evolved enzymes, largely due to product inhibition of catalytic turnover, which is a major challenge for templated dimerization and prohibitive for longer products. We present an enzyme-free DNA-based templated dimerization reaction enabled and controlled by a fuel strand that actively displaces the product from the template only once dimerization is complete, overcoming product inhibition. We systematically investigate design variants to optimise catalytic turnover, and demonstrate information propagation through the action of distinct templates that assemble specific products from the same pool of building blocks. We also show that the fuel represents an input by which the templating can be controlled, allowing the coupling of catalytic turnover to the output of upstream DNA circuitry. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/706517v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@e82eeeorg.highwire.dtl.DTLVardef@1612b85org.highwire.dtl.DTLVardef@706911org.highwire.dtl.DTLVardef@217d6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Electrochemical gradients are essential to the functioning of cells and form across membranes using active transporters. Here, we show in contrast that condensed biomolecular systems sustain significant pH gradients without any external energy input. By studying individual condensates on the micron scale using a microdroplet platform, we reveal dense phase pH shifts towards conditions of minimal electrostatic repulsion. We demonstrate that by doing so protein condensates can drive substantial alkaline and acidic gradients which are compositionally tuneable and can extend to complex architectures sustaining multiple unique pH conditions simultaneously. Through in silico characterisation of human proteomic condensate networks, we further highlight potential wide ranging electrochemical properties emerging from condensation in nature, while correlating intracellular condensate pH gradients with complex biomolecular composition. Together, the emergent nature of condensation shapes distinct pH microenvironments, thereby creating a unique regulatory mechanism to modulate biochemical activity in living systems.