ACS Bio & Med Chem Au

mRNA technology has emerged as a powerful new class of medicines. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, including treatments for cancer, metabolic disorders, cardiovascular conditions and autoimmune diseases. The 3'-polyadenylated (poly(A)) tail of mRNA is required for ribosome initiation, translation, and mRNA stability and is considered a critical quality attribute. In this study, novel direct mass spectrometry approaches were used for the analysis of both the DNA template and corresponding mRNA generated via in vitro transcription. Nucleotide resolution of the poly(A/T) sequence of the DNA template and mRNA poly(A) tail was achieved. The results show that the mRNA poly(A) tail length and heterogeneity is impacted by the heterogeneity of the DNA template, the DNA template design and RNA manufacturing conditions, including relative NTP concentrations. These results provide further important mechanistic insight into the poly(A) tail length and heterogeneity of mRNAs synthesised in vitro, including the identification of 3'-end additions of cytidine to mRNA poly(A) tails. The ability to rapidly assess DNA template quality, combined with monitoring mRNA poly(A) tail length and heterogeneity, is important as part of the characterisation of mRNA precision medicines and ensuring consistent quality of mRNA from manufacturing processes.

Cell penetrating thyclotides (CPTs) are synthetic molecules that promote highly efficient cellular uptake and endosomal escape of bioactive peptides. While peptides are valuable as medicinal agents, their translation to therapies is often limited by their inability to cross cell membranes. CPTs have a unique combination of chiral tetrahydrofurans and polar sidechains within a molecular scaffold that can be optimized to efficiently deliver peptide cargo into cells. The cellular uptake and endosomal escape of two peptides with anticancer biological activities but low bioavailabilities were remarkably improved after conjugation to a CPT. Using CPTs to overcome barriers to cellular uptake represents a new direction for the intracellular delivery of bioactive molecules, and will accelerate drug development for new medical therapies.

Non-canonical amino acids (ncAAs) are valuable tools in chemical biology and biochemistry for labeling, probing, and tracking biomolecules. ncAAs that can be recombinantly incorporated using native E. coli machinery are particularly useful because they allow for global protein incorporation and avoid complex genetic code expansion. Here, we demonstrate successful incorporation of a methionine analog, L-cyanohomoalanine (Cha), by the methionyl-tRNA synthetase of E. coli into mutant superfolder GFP (sfGFP) expressed in methionine auxotroph bacterial cultures. We compare to methionine auxotroph bacterial cultures supplemented with L-methionine (Met) or L-azidohomoalanine (Aha). In control prototrophic E. coli, bacterial growth rates are inhibited with high concentrations of Aha but not Cha. However, less sfGFP is produced in auxotrophic cells supplemented with Cha compared to Aha and Met. Thus, while Cha is non-toxic to E. coli it is incorporated less efficiently into proteins than Aha or Met. Mass spectrometry confirmed that N-terminal Cha, Aha, and Met are cleaved, as expected for the sfGFP mutants. Other sites of Cha and Aha incorporation were confirmed by mass spectrometry, with labeling efficiency varying by position. Thermal melts of purified sfGFPs demonstrate that Cha and Aha labeling does not significantly perturb the protein stability. In the future, Cha may be useful for proteome labeling by wild-type methionyl-tRNA synthetase and could be implemented in metabolic pulse-labeling of newly synthesized proteins with other methionine analogs. Additionally, the nitrile moiety of Cha may be used to perform reactions orthogonal to azide/alkyne click chemistry or could serve as a vibrational reporter of the environment.

Integrin v{beta}8 is a major activator of latent transforming growth factor-{beta} (TGF-{beta}) and an emerging therapeutic target in cancer and immune regulation. However, functional assays that directly measure v{beta}8-mediated activation of latent TGF-{beta} in a physiologically relevant context remain limited. Here, we report a co-culture cell-based reporter assay for quantitative measurement of v{beta}8-mediated activation of latent TGF-{beta}1. NIH/3T3 reporter cells were engineered to express a SMAD-responsive NanoLuc reporter, constitutive firefly luciferase for internal normalization, and cell-surface GARP-latent TGF-{beta}1. When co-cultured with v{beta}8-expressing LN-229 cells, reporter cells produced a robust signal that directly reflected localized latent TGF-{beta}1 activation. The assay demonstrated stable expression of the required biological components, reproducible signal-to-background performance, and sensitivity to benchmark v{beta}8-blocking antibodies. Inhibition studies showed potent dose-dependent blockade by an anti-v{beta}8 antibody. In contrast, pan-TGF-{beta} neutralizing antibody displayed markedly weaker apparent potency, suggesting that targeting localized v{beta}8-mediated activation is more effective than neutralizing released TGF-{beta} in this assay context. The assay also enabled screening and ranking of anti-v{beta}8 antibodies, identifying several high-potency clones, and detected v{beta}8-mediated activation of a non-cleavable latent TGF-{beta}1 mutant. This platform provides a sensitive, internally normalized, and scalable approach for mechanistic studies and therapeutic discovery targeting the v{beta}8-TGF-{beta} axis.

Thermodynamic characterization of biomolecular interactions is essential for understanding the enthalpic and entropic driving forces of molecular recognition, but established label-free techniques are limited either by bulk refractive-index sensitivity or by the lengthy thermal equilibration required to suppress it. Here, we used focal molography to investigate the temperature-dependent binding of the protein kinase A regulatory subunit (PKA-R) to cyclic AMP (cAMP) derivatives and to derive apparent thermodynamic signatures from kinetic measurements. We first validated the diffractometric readout under conditions that challenge refractometric sensors: the coherent mass density channel strongly suppressed temperature-induced bulk refractive-index effects and resolved binding in 50% human serum despite measurable non-specific adsorption, reducing the need for lengthy equilibration and buffer matching. We then combined focal molography with DNA-directed immobilization (DDI), allowing five cAMP derivatives to be presented in parallel on the same multiplexed chip and followed across five temperatures. This format yielded distinct, internally consistent apparent thermodynamic fingerprints for each derivative, separating ligands with similar affinities by their enthalpic and entropic contributions. Together, these results establish focal molography with DDI as a multiplexed workflow for comparative thermodynamic fingerprinting of biomolecular interactions at higher throughput.

Protein cages provide useful scaffolds for nanoscale engineering due to their highly ordered structures and in vivo self-assembly. These scaffolds are amendable to late-stage conjugation, enabling expansion in functionality. However, many conjugation techniques either lack site-selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Salmonella Typhimurium bacteriophage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions, via a minimal extension to the P22 coat protein C-terminus, is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction whereby two therapeutically relevant receptor targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for site-selective functionalization of large molecular assemblies like VLPs.

Fluorescence correlation spectroscopy (FCS) provides valuable information about molecular dynamics, however, experimental setup typically requires labour-intensive passivation to prevent non-specific binding of molecules to sample containers. Furthermore, precious samples can be wasted by having to use relatively high sample volumes in existing sample containers. We overcome these major issues using a simple method of sample encapsulation into water-in-oil droplets, using purified proteins and cell lysates as proof-of-concept. FCS of fluorescently labelled protein samples in the nanomolar (nM) range confirmed that water-in-oil droplets yield more accurate measurements than conventional open-chamber methods. We first optimized the droplet composition to prevent protein coating at the water-oil interface using pegylated-lipids. We then utilized FCS to accurately measure protein concentrations and diffusion speeds in nanolitre volumes. Additionally, we used fluorescence cross-correlation spectroscopy (FCCS) to measure enzymatic cleavage of substrate inside our droplet system, demonstrating the capacity of this platform to measure biological processes at the nanoscale. Overall, conducting FCS in droplets offers a cost-effective, robust, and accessible alternative for measuring molecular dynamics, with promising potential for high-throughput and resource-limited applications. TOC Image + Text O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/734730v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e0023dorg.highwire.dtl.DTLVardef@b32cb0org.highwire.dtl.DTLVardef@13ad832org.highwire.dtl.DTLVardef@47dc12_HPS_FORMAT_FIGEXP M_FIG C_FIG Conventional single-molecule fluorescence requires slow, expensive glass passivation procedures to prevent proteins adsorbing to surfaces. By encapsulating proteins in lipid-coated nanolitre water droplets, the passivation requirement is removed, enabling accurate measurement of protein dynamics in low nanolitre volumes. Water-in-oil droplets thus provide a passivation-free platform for fluorescence correlation spectroscopy.

Cytoskeletal plasticity is a defining feature of cancer progression, enabling tumor cells to adapt their morphology, mechanics, and migratory behavior during invasion and metastasis. Although actin filaments, microtubules, and intermediate filaments are known to cooperate in these processes, the molecular mechanisms coordinating their dynamics remain incompletely understood, particularly the role of post-translational modifications (PTMs). Here, we developed CytoFRET2, a multiparametric cytometry-based FRET platform that enables real-time and simultaneous monitoring of the dynamics of actin filaments, microtubules, and vimentin in living suspension cells. The system combines fluorescently tagged cytoskeletal reporters with spectral flow cytometry, allowing simultaneous high-content analysis of multiple cytoskeletal networks while overcoming autofluorescence and fluorescence interference from small molecules. Using spectral CytoFRET2, we screened a small library of epigenetic compounds targeting regulators of acetylation and methylation pathways. The screen revealed that inhibition of lysine deacetylases (KDACs) and sirtuins promoted stabilization of both microtubules and vimentin filaments, without impacting actin filament organization. In contrast, inhibition of lysine acetyltransferases (KATs), particularly with garcinol and anacardic acid, induced rapid vimentin disassembly. Mechanistically, the study reveals acetylation as a key post-translational modification regulating the dynamics of microtubules and vimentin filaments, with KAT inhibitors emerging as potent modulators of vimentin organization. Together, the findings establish spectral CytoFRET2 as a versatile platform for systematic investigation of cytoskeletal regulatory networks and therapeutic vulnerabilities in cancer.

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

The incorporation of non-canonical amino acids (ncAAs) into proteins, developed in the past 20 years, has opened new avenues with respect to protein structure, protein modification, protein-protein interaction or enzyme catalysis beyond what is possible with the 20 proteinogenic AAs. Although >300 unusual building blocks including several ncAAs have been described in nonribosomal peptides (NRPs) naturally, we aimed to further expand the scope of the underlying nonribosomal peptide synthetases (NRPS) to incorporate ncAAs beyond the naturally available ones. We have therefore systematically screened for ncAA accepting NRPS systems, applied NRPS engineering to transfer the respective ncAA-accepting parts into other NRPSs and thereby created novel peptides that were further derivatized in post-enzymatic chemical synthesis reactions directly in bacterial culture extracts. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/738027v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@90552forg.highwire.dtl.DTLVardef@1c8a5e0org.highwire.dtl.DTLVardef@2549dorg.highwire.dtl.DTLVardef@1012911_HPS_FORMAT_FIGEXP M_FIG C_FIG

Luminescence is a powerful method for detecting trace analytes and monitoring biological processes. However, most bioluminescence reagents, including luciferase and its substrates, are sensitive to temperature, limiting their useable shelf lives, and resulting in inconsistent performance. Enhancing the stability of these reagents could improve data quality, simplify workflows, and address cold chain storage issues. In this study, we demonstrate the application of the platform stabilization technology, capillary-assisted vitrification (CAV), as a tool to stabilize different luciferases and their substrates, and the application of the stabilized reagents in both in vitro and in vivo bioluminescent assays. We demonstrate that CAV-stabilized reagents can be stored and shipped ambiently, maintain consistent performance over time, and are suitable for use in cell viability quantification, tumor monitoring, in vivo imaging, microbial detection, and immunoassays. Additionally, different reagents can be co-formulated to make ready-to-use assay kits that can also be shipped and stored ambiently. Our results demonstrate that CAV stabilization is a viable alternative to traditional storage methods, with broad potential to improve bioluminescence workflows.

Enzymatic depolymerization of polyethylene terephthalate (PET) has advanced rapidly, alongside a growing volume of publicly available metagenomic data from microbial communities under sustained selective pressure from plastic exposure. Reasoning that such environments may harbor underexplored polyester-active enzymes, we developed a targeted mining workflow that screens exclusively plastic-associated datasets through multi-step bioinformatic filtering--integrating catalytic-motif screening, disulfide-topology validation, structural-similarity scoring, and phylogenetic profiling--to recover high-confidence PETase candidates. Applied to 271 plastic-associated metagenomes, the pipeline yielded 21 non-redundant candidates, several of which combine the Type I catalytic motif (GHSMGGGG) with Type II-like extended loops and secondary disulfide bonds. Two candidates were experimentally confirmed as PET hydrolases; the more active, PET-KR1, is a thermostable enzyme (Tm = 66.5 {degrees}C) that depolymerizes PET across a broad temperature range, with markedly higher productivity on powdered than on film substrate. PET-KR1 achieved optimal depolymerization at 50 {degrees}C, yet at 60-65 {degrees}C, where total yields declined, the product pool was more strongly enriched in the terminal monomer TPA, suggesting that thermostability and substrate accessibility are the primary targets for further engineering. Molecular dynamics simulations revealed a conserved hydrophobic binding network around the catalytic serine, consistent with established PETase substrate-recognition modes, and rational disulfide engineering raised the melting temperature by 3.5 {degrees}C, confirming amenability to further optimization. Overall, PET-KR1 expands the scaffold space available for PETase engineering, while the discovery workflow, built entirely on publicly available tools and open-access data, provides a reproducible strategy for metagenomic mining of novel PET-degrading enzymes toward biocatalytic PET recycling.

Precisely modifying proteins at multiple sites in their native, folded structures offers unique opportunities to answer molecular and cellular-level biological questions. Here, we present a genetic code expansion strategy for site-specific integration of a fluorophore-quencher pair comprising two non-canonical amino acids--acridonylalanine (Acd) and methyltetrazinyl phenylalanine (Tet) -- into a protein expressed in E. coli. The Acd and Tet pair requires no post-translational labeling, and quenching can be switched off by biorthogonal or photochemical reactions of Tet for convenient internal control experiments. Mechanistic studies based on Stern-Volmer quenching, fluorescence lifetime measurements, and "proline ruler" peptides established the distance dependence of quenching. As proof-of-concept, we applied this strategy to study: 1) calmodulin, a calcium-sensing protein, 2) RecA, a DNA damage sensor in bacteria, and 3) LexA, a transcriptional repressor whose activation by RecA governs acquired antibiotic resistance in bacteria. Using these proteins, we demonstrate that dual Acd/Tet labeling provides molecular-level insights into protein dynamics, enables high-throughput drug screening, and advances tools for studying protein structure-function relationships.

Protein tyrosine phosphorylation is critical for cellular function, and aberrant phosphorylation is tied to a wide range of human diseases. Identifying the substrates of protein tyrosine phosphatases, the enzymes that erase this modification, is critical to understanding human biology and disease states. The state-of-the-art method for tyrosine phosphatase substrate identification requires the use of mutations that modestly increase the lifetime of enzyme-substrate complexes by kill catalytic activity. While these substrate-trapping mutants are useful tools, they work best for high-affinity or abundant substrates that remain phosphatase-bound through cell lysis and enrichment. Here, we use site-specific photo-crosslinking to covalently capture the substrates of tyrosine phosphatases in situ. We identify eight different positions around the active site of the phosphatase PTP1B where photo-crosslinker amino acids can be incorporated via amber codon suppression without dramatically disrupting catalytic activity. We then conduct photo-crosslinking experiments in mammalian cells and identify crosslinked proteins by mass spectrometry proteomics, revealing that our approach can capture known PTP1B interactors and substrates. We then show that PTP1B photo-crosslinking in situ is sensitive to enzyme localization and identify new PTP1B substrates that regulate contacts between the endoplasmic reticulum and plasma membrane. We also demonstrate that photo-crosslinking can capture signal-dependent interactions. For example, we observe PTP1B crosslinking to the epidermal growth factor (EGF) receptor, a known substrate, in an EGF stimulation-dependent manner, and we identify other potential EGF-dependent substrates. Overall, our approach reveals previously unknown roles of PTP1B in signaling systems and could be readily extended to other tyrosine phosphatases in the same family.

The function of a protein depends not only on its sequence but on post-translational modifications and folding that produce functionally distinct proteoforms. Single-molecule methods for protein identification, such as nanopore sequencing, typically require denaturation or proteolysis, sacrificing conformational information that contributes to proteoform diversity. Here, we identify intact, folded proteins by recording an optical fingerprint of their local surface chemistry using a single covalent label. The signal is produced by a spontaneously blinking fluorophore attached to the protein through established bioconjugation reactions. The thermodynamics and kinetics of its switching between a fluorescent and a dark state are influenced by the immediate protein environment in a chemically interpretable manner. Further discriminative information can be extracted using deep learning to achieve excellent identification accuracy. Using this approach, we distinguish different proteins, different pockets of the same protein, and the presence of a single post-translational modification, in each case tracing the classification back to a distinct physicochemical mechanism. These results establish single-molecule fluorescence blinking as both a protein fingerprinting method and a probe of local chemistry on the surface of folded proteins.

Despite its importance in cellular signaling and protein fate, the detection of protein ubiquitination in proteomics experiments presents many challenges for researchers. Importantly, current techniques that often rely on antibodies specific for lysine sidechain modifications may miss non-canonical ubiquitination sites in experiments. We envisioned a strategy that uses sortase, a bacterial transpeptidase enzyme, to selectively modify ubiquitination sites with a Biotin tag for enrichment and downstream proteomics experiments. In this work, we demonstrate our ability to selectively modify N-terminal diglycine remnants in digested proteins with a Biotin-modified peptide, enabling downstream enrichment of previously ubiquitinated proteins. We show this proof of concept on several recombinant proteins, revealing a site of autoubiquitination in the E2 conjugating enzyme Ubc13. We show that elution of the enriched peptides can be achieved by using common guanidinium elutions or by leveraging the reversibility of sortase. Finally, we include a bifunctional peptide that is labile to trypsinization to better streamline this strategy for downstream proteomics approaches. We envision that this approach will provide an accessible strategy for the detection of ubiquitinated proteins in proteomics experiments, with the goal of enabling researchers to better detect noncanonical protein ubiquitination.

Therapies to activate the STING immune response pathway represent promising potential anticancer treatments. However, the native STING activating molecule, 2',3'-cGAMP, is a poor drug candidate due to its susceptibility to nuclease degradation and its relatively poor cell uptake. In this study, we present a nanoscale delivery vehicle based on the bacteriophage MS2 virus-like particle that can both protect cGAMP and deliver it into cells to access and bind cytosolic STING. MS2-delivered cGAMP achieved greatly increased STING activation potency relative to both free cGAMP and a nuclease-resistant synthetic cGAMP analog. In an in vivo murine colon carcinoma model, MS2-cGAMP elicited significant and prolonged antitumor activity in a STING-dependent manner at 50-fold lower concentrations relative to free cGAMP and synthetic analogs. These results demonstrate that MS2 delivery of cGAMP can yield a highly potent STING agonist immunotherapy with in vivo anticancer activity.

Poor aqueous solubility remains a major obstacle to the translational development of targeted anticancer compounds. VS1, a first-in-class inhibitor of the cholesterol-transfer protein STARD3, has emerged as a promising chemosensitizing agent in colorectal cancer (CRC), but its clinical applicability is limited by its poor water solubility. Here, we combine structural biology, nanotechnology, and functional pharmacology to establish STARD3 inhibition as a delivery-enabled strategy to potentiate fluoropyrimidine therapy. To define the molecular basis of STARD3 inhibition, we solved the crystal structure of VS1 bound to the STARD3 ligand-binding domain at 2.1 [A] resolution, revealing direct occupation of the sterol-binding cavity. Molecular dynamics simulations confirmed a stable binding mode and identified the {Omega}1 loop as a dynamic gate regulating ligand binding and dissociation. To overcome the formulation barrier of VS1, we engineered carrier-free, albumin-coated nanocrystals through sonication-assisted nanocrystallization followed by surfactant exchange with human serum albumin. The resulting rod-shaped nanocrystals displayed nanometric size, narrow size distribution, sustained release, and improved aqueous dispersibility, increasing the apparent solubility of VS1 by more than 14-fold while preserving its molecular integrity and crystallinity. Biologically, VS1 selectively potentiated 5-fluorouracil (5-FU) in CRC cells, with synergistic effects restricted to 5-FU-sensitive models and associated with enhanced reactive oxygen species accumulation. Albumin-coated formulation retained the chemosensitizing activity of the free compound. In HCT-116 xenografts, combined treatment with albumin-coated VS1 nanocrystals and 5-FU significantly reduced tumor growth, prolonged tumor doubling time, and increased intratumoral necrosis without exacerbating systemic toxicity. Together, these findings establish that albumin-coated nanocrystals can overcome the delivery limitations of an insoluble STARD3 inhibitor and provide a formulation-enabled strategy to enhance fluoropyrimidine therapy in colorectal cancer.

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite's ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin-streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied this method to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.

Engineering synthetic cells where metabolism directly controls gene expression is one of the greatest challenges in synthetic biology. This coupling between metabolic activity and protein synthesis is also thought to have been a vital step in the evolution of the earliest cellular life. This fundamental process is essential for developing minimal, self-sustaining cells, both engineered for biotechnology applications and as models explaining the origins of life. Here, we present a programmable cell-free platform that links metabolic activity to translation under defined resource limitations. Using an engineered amino-acid-dependent cell-free translation system, we introduced a tunable metabolic bottleneck (depleting tyrosine). This enabled imposing a controlled metabolic constraint on protein synthesis. To alleviate this constraint, we then incorporated phenylalanine hydroxylase (PAH) as a minimal module for tyrosine synthesis. The PAH-driven tyrosine synthesis established a system in which protein expression is directly controlled by amino acid biosynthesis. This relationship was recapitulated in liposome compartments, resulting in three distinct synthetic cell populations. The metabolically active population had significantly higher fitness in protein production. Overall, this work establishes an experimentally tractable platform to investigate how primitive cells may have evolved internal metabolic capabilities, and it represents a foundational step toward constructing more autonomous and self-regulating synthetic cells.