Nature Chemistry

The declining cost of green hydrogen--projected below 1.5 USD/kg by 2030--opens new avenues for its use beyond fuel cells and industrial heating. Here we demonstrate that H2 can serve as a stoichiometric electron donor for cell-free enzymatic cofactor regeneration, coupling H2 oxidation to NADPH production and driving the complete bioconversion of pyruvate to lactate. A partially purified enzyme ensemble from Escherichia coli overexpressing Clostridium pasteurianum ferredoxin, augmented with [FeFe]-hydrogenase CpII, delivers NADP+ reduction rates of 103 M min-1 (27-fold enhancement) with superlinear dependence on H2 partial pressure. Reconstitution from purified components (CpI or CpII, CpFd, AnFNR, LDH) uncovers a redox-potential-dependent lag phase: the NADPH/NADP+ ratio must exceed 0.85 before pyruvate reduction becomes thermodynamically spontaneous, after which the rate accelerates exponentially. These results position hydrogen-driven cofactor regeneration as a scalable, byproduct-free platform for reductive biotransformations powered by renewable H2.

RNA is crucial to all extant biology and thought to have played an essential role in lifes origins. However, hydrolysis of RNA under common environmental conditions such as heating, drying, and exposure to divalent cations presents challenges for its persistence and effectiveness in prebiotic contexts. RNAs with less stable secondary and tertiary structures are especially susceptible to degradation. In living cells, sequestration in biopolymer-rich membraneless organelles helps shield RNA from damage and enzymatic degradation. Here, we report that compartmentalization into polyelectrolyte-based complex coacervate droplets protects RNA from hydrolysis during wet-dry cycling at elevated temperatures in the presence of Mg2+. This protection can be understood in terms of the coacervate microenvironment under varied salt concentrations and how it changes during sample drying. After gaining insight into this prebiotically-plausible mechanism using an unstructured model RNA, we demonstrate coacervate-based protection of a ligase ribozyme, supporting the functional relevance of the protection.

Sequence heterogeneity is a defining feature of cellular biomolecular condensates, yet how competing interaction motifs encode their thermodynamic stability, internal organization, and dynamics remains poorly understood. Here, we systematically tune the hydrophobicity mismatch between intrinsically disordered peptide pairs to establish sequence hydrophobicity as a programmable determinant of heterotypic condensate behaviour. We show that heterotypic condensates are thermodynamically more stable than homotypic ones having same average hydrophobicity through the cooperative interplay of short-range hydrophobic and long-range electrostatic interactions. Increasing hydrophobicity mismatch drives a composition-dependent transition from homogeneous condensates to core-shell architectures accompanied by pronounced spatial and dynamical heterogeneity, whereas reducing sequence disparity restores homogeneous organization and nearly uniform dynamics. Our results establish a direct molecular link between sequence chemistry, phase stability, condensate architecture, and transport dynamics, providing predictive design principles for engineering synthetic biomolecular condensates with programmable organization and material properties. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/743201v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@12e7fborg.highwire.dtl.DTLVardef@13c31a0org.highwire.dtl.DTLVardef@de3453org.highwire.dtl.DTLVardef@3d4600_HPS_FORMAT_FIGEXP M_FIG C_FIG

Early ribonucleoprotein systems likely required mechanisms to concentrate and organize RNA. Modern ribosomes use Mg2+ and evolved Lys/Arg-rich protein extensions to stabilize their RNA backbone. Before templated synthesis, however, peptide formation likely generated heterogeneous sequence populations rather than reproducible ribosomal sequences. Here, we compare Mg2+, ribosomal peptides, and statistical peptide libraries in organizing a 136-nucleotide model of the peptidyl transferase center (sPTC). Mg2+ condenses sPTC only at a large excess of positive charge following thermal annealing, producing largely arrested libraries. Acidic conditions further promote condensation. Ribosomal peptides instead promote coacervation near charge stoichiometry and form droplets that readily fuse. Several statistical peptide libraries also coacervate with sPTC, despite comprising heterogenous mixtures rather than a single defined sequence. Increasing mean positive charge favors condensation, but Lys/Arg-containing libraries undergo liquid-liquid phase separation more readily and across broader conditions than matched libraries containing the prebiotically plausible diaminopropionic acid (Dpr) and diaminobutyric acid (Dab). Atomistic computer simulations implicate Arg as a major source of this difference, as it can form more numerous and longer-lived hydrogen bonds with RNA, while competitive partitioning experiments show preferential condensate recruitment in the order Arg > Lys > Dab > Dpr. Together, our findings show that peptide-RNA coacervation can be triggered collectively by statistical peptide ensembles, providing a plausible route to protoribosomal organization without peptide sequence-specific optimization. Cationic chemistry shapes condensate formation and material properties, suggesting that amino acid alphabet formation could have broadened the conditions supporting liquid-like protoribosomal assemblies.

Isopeptide bonds are amide bonds between amino acid side chains that can form autocatalytically, notably in the pili of Gram-positive bacteria. Here, we design de novo proteins that form both intramolecular and intermolecular isopeptide bonds entirely autocatalytically. We report over 50 designs that form isopeptide bonds, validated by mass spectrometry and 5 crystal structures. We redesign these constructs as split proteins that form a covalent intermolecular isopeptide crosslink when combined. These split designs are orthogonal to the existing isopeptide-based SpyTag/Catcher system, and their formation can be regulated by temperature, providing control over the timing of crosslinking in protein assemblies. We extend these designs to create rigid domain crosslinks that enable the construction of large well ordered symmetric rings of up to 215 kDa that are irreversibly covalently crosslinked by multiple isopeptide bonds into a single molecule. Our results provide insight into the determinants of isopeptide bond formation, considerably expand the set of isopeptide bond crosslinking systems, and establish a framework to construct fully covalent rigid protein assemblies.

Biomolecular condensates, which regulate diverse cellular processes, exhibit distinct electric potential profiles. This potential gradient between the dilute and the dense phases serves as the underlying driving force mediating the unique microenvironment and electrochemical activity of condensates. However, the molecular principles encoding the electric potential profiles of condensates remain unclear. In this study, we show that molecular asymmetry is a unifying origin of electric polarization in condensates. Asymmetric protein-cation and protein-anion affinities alone generate an interfacial electric double layer and a finite potential even in condensates formed by charge-free proteins. The sign of potential gradient follows the direction of the affinity bias, and the magnitude collapses onto a single linear function of dense-phase protein volume fraction across changes in chain length, interaction strength and salt concentration. Further, chain termini preferentially occupy the condensate interface, so charges positioned asymmetrically with respect to the termini create spatial charge separation even in neutral polyampholytes. These interaction-encoded and sequence architecture-encoded asymmetries can reinforce, screen or reverse one another, allowing the magnitude and polarity of the interphase potential to be tuned through sequence design or solvent environments.

The ability to map protein interactomes and organelle proteomes is foundational for achieving a molecular understanding of living cells. Proximity labeling (PL) provides a powerful strategy for this, but existing enzymes and photocatalysts are limited by their spatial resolution, reliance on biotin, and/or in vivo compatibility. Here we report FlexID, an engineered promiscuous ligase that catalyzes the rapid attachment of diverse small-molecule probes to proximal endogenous proteins. Critically, FlexID operates through a zero-radius, direct-contact mechanism, offering superior spatial precision compared to existing PL tools. We engineered FlexID by combining the strengths of sequence- and structure-trained computational models to enhance its catalytic activity and structural stability. Biophysical analysis revealed that specific conformational changes in FlexID improve its ability to recognize diverse target proteins while simultaneously preventing the premature release of the reactive intermediate. We demonstrate FlexID's versatility through in vivo proximity labeling, comprehensive organelle proteome mapping, and a high-throughput, fluorescence-based screen for molecular glues. Our work shows that computational methods can be harnessed to create mechanistically distinct PL enzymes and establishes FlexID as a flexible, high-resolution tool for mapping protein interactions and proteomes in living cells.

Catalyst-controlled stereodivergent synthesis of acyclic molecules bearing multiple stereogenic centers remains a longstanding challenge in asymmetric catalysis, particularly through intermolecular radical C-C coupling. Here, we report the directed evolution of pyridoxal 5'-phosphate (PLP)-dependent biocatalysts for the stereodivergent synthesis of non canonical amino acids bearing either skipped , {gamma}-stereochemical dyads or contiguous , {beta}, {gamma}-stereochemical triads. Starting from a thermophilic tryptophan synthase {beta}-subunit, protein engineering generated complementary enzyme variants that selectively construct three of the four possible , {gamma}-stereoisomers through biocatalyst-controlled reversal of both remote {gamma}-stereochemistry and proximal -stereochemistry. These engineered enzymes catalyzed intermolecular radical C-C bond formation with a broad range of secondary alkyltrifluoroborates, affording , {gamma}-disubstituted amino acids in excellent yields and stereoselectivities. Furthermore, directed evolution enabled efficient {beta}-dehydroxylative radical coupling of the non-native threonine-derived aminoacrylate intermediate, providing amino acids bearing contiguous , {beta}, {gamma}-stereochemical triads with up to 96:4:<0.1:<0.1 d.r. and >99:1 e.r.. To further demonstrate the synthetic utility of this platform, the stereochemically enriched amino acids were transformed into valuable {gamma}-chiral amines through engineered PLP dependent tryptophan decarboxylases, establishing a dual PLP enzyme based biocatalytic cascade for the enantiodivergent synthesis of privileged amine scaffolds. Molecular dynamics simulations reveal that enzyme controlled {gamma}-stereoselectivity originates from active-site remodeling that modulates {pi}-{pi} interactions and steric contacts governing the facial selectivity of the radical addition step. This work expands the synthetic capabilities of radical biocatalysis by enabling catalyst-controlled stereodivergent assembly of complex acyclic stereochemical arrays and establishes engineered PLP enzymes as versatile catalysts for the synthesis of stereochemically complex amino acids and chiral amines.

Biomolecular condensation emerges from networks of multivalent interactions that respond collectively to molecular composition and environmental conditions. Here, we establish a sequential Design of Experiments (DoE) framework to systematically and efficiently characterise multidimensional condensation landscapes. Applied to three RNA-binding condensate proteins - Dhh1, Ded1 and PGL-3 - the workflow revealed unexpected protein-specific interactions between biophysical and biochemical variables, including RNA, pH and ionic strength, and uncovered context-dependent switch-like behaviours. Extending the framework to protein mixtures revealed how proteins affected each other's condensation and whether they formed condensate-within-condensate structures under different experimental conditions. Finally, an unexpected response identified by DoE prompted mechanistic analysis of Dhh1, revealing an ATP-independent RNA-binding surface that promotes efficient condensation in vitro and contributes to P-body formation in cells. Sequential DoE therefore provides an experimentally accessible strategy for uncovering context-dependent condensate regulation and converting multidimensional response patterns into mechanistic hypotheses.

Molecular glue degraders (MGD), a new drug modality that utilizes the natural proteasomal degradation machinery in cells for target protein degradation, is drawing great attraction from the pharmaceutical industry in recent years. However, the pace of MGD molecule discovery can be largely limited by the low throughput of conventional chemical synthesis and tedious purification processes. It is long-awaited to develop a new screening strategy to release the potentials of high-throughput chemical synthesis while organically integrating compound library construction with assay testing. Here, by leveraging the mild reaction conditions of amide coupling, we demonstrate the implementation of "Direct-to-Biology" (D2B) strategy for Cereblon (CRBN)-targeting MGD discovery with in-plate microscale high-throughput library construction directly followed by cellular HiBiT-based target protein degradation assay screening without purification. Our results indicate that the crude D2B compound mixtures can show comparable efficacy and potency as their purified counterparts. And by systematically integrating chemistry with biology, we can significantly accelerate the pace of MGD discovery.

Azobenzene photolipids are versatile actuators of membrane mechanics and protein function, yet long-wavelength control and leaflet-selective perturbations remain difficult to impose. Here, we address both limitations. First, we show that red-light excitation of a lipidated Nile Blue derivative (NB-lipid) catalyzes rapid and reversible cis[-&gt;]trans photoisomerization of co-localized azobenzene photolipids without requiring covalent conjugation. Second, we demonstrate that photocatalysis is predominantly intraleaflet, enabling leaflet-selective actuation by incorporating the membrane-targeted chromophore into only one bilayer leaflet. This enables on-demand and rapid writing of transbilayer mechanical asymmetry, which we track via lock-in capacitance signatures. Critically, symmetric and asymmetric leaflet mechanical perturbations can be evoked within the same membrane by illuminating with blue or red light, respectively. Photocatalysis of electron-rich azobenzenes is anticipated to proceed via the singlet excited state of NB-lipid and thus avoids triplet-state phototoxicity. We underscore the biocompatibility of catalysis by demonstrating the effect of leaflet-specific catalyzed perturbations on the ROS-sensitive mechanosensitive peptide ion channel gramicidin A. Our results establish a novel tool for dissecting the role of dynamic leaflet-specific mechanical changes in membrane remodeling and protein function.

Enzymes catalyze multistep reactions by stabilizing successive transition states within well organized, yet dynamic active sites. However, computational enzyme design typically targets a single transition state using rigid active-site models. Here, we introduce multistate enzyme design, which uses conformational ensembles to optimize active sites across an entire reaction coordinate. Applied to a de novo Morita--Baylis--Hillmanase, multistate enzyme design outperformed conventional single-state design, with the most active variant achieving >100-fold higher bi-substrate catalytic efficiency and surpassing an extensively optimized enzyme from directed evolution in both efficiency and enantioselectivity. Structural and kinetic analyses revealed that multistate design preserved catalytic preorganization and conformational plasticity, distributed stabilization across the reaction coordinate and avoided kinetic bottlenecks created by single-state optimization. By contrast, single-state design compromised preorganization, destabilized upstream states and shifted rate limitation away from the targeted transition state. Multistate enzyme design provides a framework for designing catalytic landscapes rather than static active sites, opening a route to efficient de novo enzymes for complex multistep chemistry.

Many biological proteins function by changing shape upon small-molecule binding. Here, we present a general strategy for designing de novo proteins that undergo small-molecule-induced conformational change. Our approach converts a preorganized small-molecule binding protein into a ligand-responsive shape-changer by adding a mobile lid domain that closes behind the ligand upon binding. Using this strategy, we converted an exatecan-binding protein into a drug-induced conformational switch. The lidded proteins showed considerably stronger binding affinity in the sub-nanomolar regime, 100-fold greater specificity to exatecan over a similar molecule, and ligand residence times up to several months, with tunable binding kinetics. We turned one design into a genetically encodable fluorescent biosensor of the drug, enabling potential clinical applications. Our results open the door to programming complex molecular function using vast chemical space.

Heavy water (D2O) is widely used in biomolecular spectroscopy and imaging, often under the assumption that it is an inert replacement for H2O. However, D2O differs subtly in hydrogen-bonding, viscosity, and dielectric properties, which can alter biomolecular interactions and self-assembly. Here, we test how solvent isotope substitution modulates protein/peptide phase separation and amyloid formation in multiple intrinsically disordered systems. Using turbidity-based phase diagrams and microscopy, we quantify how D2O shifts protein-RNA complex coacervation boundaries and alters condensate morphology. Droplet recovery measurements indicate significant solvent-dependent changes in condensate material properties. We further evaluate amyloid formation kinetics, in the presence or absence of a cofactor, supported by orthogonal structural characterization, and assess the functional consequences of tau fibrils using a tau biosensor seeding assay with explicitly defined seed delivery conditions. Together, these results show that D2O can systematically bias liquid-liquid phase separation and aggregation readouts and should be treated as an active experimental variable rather than a neutral solvent substitute.

Optically addressable spins, traditionally studied in semiconductors and, more recently, in (bio)chemical systems, are central to quantum technologies, yet existing platforms lack scalable and accessible site-specific programmability. Here, we show that DNA can serve as a functional nanoscale scaffold for optically addressable spin systems. By incorporating flavin chromophores into synthetic oligonucleotides, we generate spin-correlated radical pairs (SCRPs) that can be manipulated by radiofrequency (RF) fields and read out using optically detected magnetic resonance (ODMR). Pulsed ODMR resolves spin dynamics on sub-microsecond timescales under ambient conditions, while DNA sequence design enables atomically precise tuning of both the ODMR response and the associated spin chemistry with single-base resolution. DNA secondary structure provides an additional layer of functionality: duplex formation inverts the pulsed ODMR contrast, indicating a switch in the spin multiplicity of the SCRP precursor. The synthetic accessibility and chemical programmability of oligo-nucleotides as hosts for optically addressable spins are demonstrated through a series of proof-of-concept applications, including sensing, programmable SCRP positioning, and spin-enhanced molecular beacons. Our results establish DNA as a versatile scaffold for engineered spin systems, providing a platform for future applications ranging from quantum sensing and programmable spin arrays to bioimaging and RF-controlled molecular switches for gene regulation.

The fosfazinomycins are phosphonate natural products with antifungal activity. Their main scaffold is composed of a phosphonate moiety attached to the carboxylate of arginine via a hydrazine linkage. Previous studies have elucidated a convergent biosynthetic pathway that independently assembles phosphonate and hydrazine synthons, but how these building blocks are then connected could not be determined. In this work, we provide the final missing steps in fosfazinomycin biosynthesis by revealing an unexpected engagement of biosynthetic machinery that is typically involved in ribosomally synthesized and post-translationally modified peptides (RiPPs). An asparagine synthetase-like (AS-like) enzyme catalyzes the installation of hydrazine onto the carboxylate of Arg at the C-terminus of a short ribosomally synthesized precursor peptide. The terminal nitrogen of the resulting peptide hydrazide is methylated, and the phosphonate moiety is activated to a triphosphate-like intermediate through two separate kinase catalyzed phosphorylation steps. A nucleotidyl transferase then catalyzes the ligation of the two fragments to generate the mature fosfazinomycin scaffold on a peptide. Aminopeptidase cleavage of this peptide then yields fosfazinomycin B (fosB), which serves as substrate for a valinyl-tRNA dependent reaction to afford fosfazinomycin A (fosA). This work demonstrates an unprecedented example of the convergence of RiPP and phosphonate biosynthetic logic, an enzymatic route to peptide hydrazides that are widely used in peptide ligation chemistry, and an unusual activation sequence for conjugation of phosphonates.

The experimentally observable dynamical landscape of biomolecules is fundamentally shaped by rapid thermal motions of the surrounding aqueous environment. Although lowering temperature could expand this observable landscape, the liquid-solid phase transition of water has long prevented real-time single-molecule measurements into deeply subzero aqueous environments. Here we show that nanoconfinement within a solid-state nanopore overcomes the fundamental limitation imposed by bulk water freezing, spontaneously stabilizing a persistent liquid-in-ice environment that remains electrically accessible despite surrounding electrolyte crystallization. This aqueous environment creates a time-stretched dynamical regime, extending molecular translocation timescales by up to [~]400-fold and revealing previously inaccessible single-molecule dynamics. These findings establish a new low-temperature aqueous regime for real-time single-molecule measurements, opening new opportunities to investigate biomolecular dynamics across previously inaccessible timescales and extreme aqueous environments.

Enzyme engineering aims to tailor natural enzymes for industrial and therapeutic applications, yet physically grounded rational design has been limited by a trade-off between accuracy and cost, leaving the field heavily dependent on expert intuition. Here we present a scalable physics-based framework that combines field-aware machine learning with molecular mechanics to capture enzyme electrostatics at quantum-mechanical accuracy while enabling efficient, atomistic exploration of reaction free-energy landscapes. Coupled with microkinetic modelling, the framework translates molecular free-energy landscapes into catalytic rates and selectivity across competing, multistep reaction pathways. Applied to a newly engineered oxidative amidase (OxiAm), the framework predicts catalytic rate constants with near-experimental accuracy, quantitatively resolves the selectivity between hydrolysis and aminolysis, and generalizes across substrates, mutations and enzyme homologues. Transition-state ensemble analysis further reveals the reaction mechanism and guides the design of enzyme variants for pharmaceutical synthesis. By bringing chemical accuracy and high-throughput sampling to enzyme catalysis, this approach shifts rational design from static, empirical practice toward dynamic, free-energy-driven design, and should accelerate the engineering of biocatalysts.

Messenger RNA (mRNA) therapeutics have transformed vaccination and protein replacement strategies, yet efforts to improve their performance have focused largely on sequence engineering, nucleotide modification, and delivery vehicles. Here we show that mRNA function can be controlled by rational design of higher-order RNA architectures. We develop self-assembling mRNA origami (mRNA-OG), a class of unimolecular RNA nanostructures that encode protein-coding sequences within higher-order, programmable, and compact nucleic acid architectures. Using computational design and experimental validation, we demonstrate that mRNA-OG folds into well-defined nanostructures while remaining translationally competent in mammalian cells. Although folded mRNA-OG recruits ribosomes comparably to unfolded constructs, it produces lower protein output, indicating that RNA architecture can directly influence translational efficiency. The compact geometry of mRNA-OG also enhances encapsulation by cationic lipid delivery systems, suggesting a structural route to improved cargo packaging. Beyond its effects on translation, mRNA architecture modulates innate immune recognition. In primary human dendritic cells, folded and unfolded mRNA-OG elicit distinct cytokine programs and differential activation of stress-response pathways, including a modest induction of the integrated stress response that is not fully explained by canonical PKR signaling. Our results establish programmable structure as a new design parameter for mRNA therapeutics that can affect its functionality, delivery properties, and immune sensing.

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.