Angewandte Chemie International Edition

Glycoside hydrolases (GHs) play central roles in carbohydrate metabolism and are widely exploited for industrial and biomedical applications. However, they are often not optimal for applications due to their constrained function and strict stereochemical specificity, necessitating the discovery and optimization of distinct enzymes for each glycosidic configuration. Members of glycoside hydrolase family 1 (GH1) are archetypal retaining {beta}-glycosidases, while -specific activity is rare within this family. Here, I demonstrate that a retaining GH1 enzyme can be engineered to hydrolyze both {beta}- and -configured substrates without altering its canonical catalytic residues. Using a well-characterized {beta}-glycosidase and computational protein design strategies targeting second-shell residues surrounding the active site, a bifunctional {beta}-/-glycosidase containing 45 mutations was generated. The engineered variant acquired the ability to hydrolyze the -configured substrate 4-nitrophenyl--D-glucopyranoside while retaining activity toward the originals {beta}-substrates, with reduced catalytic efficiency and thermostability. Structural modeling and docking analyses reveal that the engineered enzyme preserves the original fold and accommodates substrates within the catalytic pocket in a similar manner to the wild type. These findings provide direct evidence that stereochemical constraint in retaining GH is more flexible than previously appreciated and can be modulated through targeted engineering.

Modern drug discovery demands efficient strategies for generating structurally diverse compound libraries. Skeletal editing--a transformative paradigm enabling precise atom-level modifications within molecular frameworks, offers a sustainable alternative to traditional synthetic routes. While carbene insertion-mediated approaches have dominated single-carbon insertion strategies, current methodologies are limited by their reliance on hazardous, unstable carbene precursors and harsh reaction conditions. Herein, we report a multicopper oxidase (MCO)-catalyzed skeletal editing that enables the direct, one-step transformation of phenolic and indole derivatives into functionalized tropones and quinoline analogues through exogenous single-carbon insertion. This platform employs stable and safe nitroalkanes as carbon sources and O2 as the sole terminal oxidant. It accommodates a broad substrate scope and yields products with superior antibacterial activity against to multidrug-resistant strains relative to their parent compounds. This work introduces the first biocatalytic platform for exogenous single-carbon insertion skeletal editing. This sustainable and scalable strategy overcomes key limitations of synthetic approaches, offering efficient skeletal remolding and rapid expansion of bioactive compound libraries. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/714988v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@ed9336org.highwire.dtl.DTLVardef@15beeeaorg.highwire.dtl.DTLVardef@a26525org.highwire.dtl.DTLVardef@19e7707_HPS_FORMAT_FIGEXP M_FIG C_FIG

Conformational dynamics play a central role in enzyme function by controlling substrate access and productive binding. Yet mutations that beneficially modulate these properties are difficult to identify. Here, we used ultrahigh-throughput fluorescence-activated droplet sorting (FADS) with a bulky fluorogenic substrate derived from coumarin (COU-3) to impose steric selection pressure on the haloalkane dehalogenase LinB. Screening a focused library yielded five single substitutions located 11.5-15.5 [A] from the catalytic centre. Variant I138N showed a fourfold increase in catalytic efficiency toward COU-3 through reduced KM and increased kcat, associated with increased cap-domain flexibility and facilitated substrate entry. In contrast, variant P208S markedly reduced substrate inhibition and shifted specificity toward bulkier iodinated haloalkanes by reshaping its tunnel environment. Integrated kinetic and structural analyses revealed that screening with bulky substrates directs selection toward distal regions controlling substrate access and unproductive binding. These findings demonstrate that ultrahigh-throughput FADS can reveal dynamic mechanisms of enzyme adaptation that remain difficult to predict by rational design. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/713925v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@782038org.highwire.dtl.DTLVardef@8b43f3org.highwire.dtl.DTLVardef@11a403eorg.highwire.dtl.DTLVardef@6fcaea_HPS_FORMAT_FIGEXP M_FIG C_FIG

Progress in understanding protein-ligand interactions is revolutionizing drug design, especially for G protein-coupled receptors (GPCRs), which are targets for 35% of marketed drugs.1 The dopamine D2 receptor (D2R) represents a key drug target in schizophrenia and Parkinsons disease.2 While structural studies have clarified its interactions with classical ligands, the behavior of atypical, non-basic ligands like D2AAK2 remains unclear. Notably, D2AAK2 shows strong selectivity for D2R over the closely related D3R, despite identical binding pocket composition. Here, we present a cryo-EM structure of D2AAK2 bound to D2R, showing that aspartate 3.32 serves as the main anchoring point, even though the compound lacks a basic nitrogen atom. Using enhanced sampling molecular dynamics simulations and experimental approaches, we uncover a complex binding energy landscape. Simulations suggest that D2AAK2 receptor subtype selectivity between identical binding sites arises from different energy barriers for their conformational changes. Non-basic ligands offer advantages such as better brain penetration and improved pharmacokinetics.3,4 This study provides the first structural insights into a non-basic ligand targeting D2R, paving the way for developing more effective, selective drugs.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/707154v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e81c3borg.highwire.dtl.DTLVardef@1958c6borg.highwire.dtl.DTLVardef@1360015org.highwire.dtl.DTLVardef@3f9388_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Protein-protein interactions (PPIs) mediated by extracellular ligands remain challenging targets for small molecule intervention due to their large and dynamic interfaces. The interaction between SLIT2 and its receptor ROBO1 plays a critical role in cell migration and tumor progression, yet remains largely unexplored. Here, we report the discovery and optimization of small molecule inhibitors of the SLIT2/ROBO1 interaction enabled by DNA-encoded library (DEL) screening. Affinity selection against SLIT2 identified four structurally diverse hit compounds, which were subsequently validated using orthogonal biophysical assays. Among these, one hit exhibited measurable SLIT2 binding and functional inhibition of the SLIT2/ROBO1 interaction in a time-resolved FRET assay. Guided by physicochemical considerations, a solubility-optimized analog was designed, resulting in a [~]50-fold improvement in binding affinity and an [~]9-fold enhancement in functional potency. Molecular dynamics simulations and induced-fit docking revealed a stable binding mode within the SLIT2 LRR2 domain and suggested that a benzothiophene substituent was dispensable for target engagement. Fragment-based experimental validation confirmed this prediction, leading to the identification of a minimal azaindole-based pharmacophore that retained nanomolar binding affinity. Collectively, this study demonstrates how DEL-enabled hit discovery combined with rational optimization and fragment deconstruction can yield potent small molecule modulators of a challenging extracellular PPI, providing a foundation for further development of SLIT2/ROBO1 pathway inhibitors.

Mutant KRAS is a potent oncogene, serving as a tumor driver in many solid human cancers. Current small-molecule inhibitors target the highly conserved G-domain, but to gain further mechanistic insight into the roles of different isoforms, we investigated the strategy of sterically shielding the unstructured hypervariable regions (HVRs). KRAS HVRs undergo a series of post-translational modifications that enable intracellular trafficking and membrane attachment. Previous attempts to drug KRAS by preventing its post-translational modification, based on inhibition of the involved prenylation enzymes have been largely unsuccessful. In this study, we explored the property of Designed Armadillo Repeat Proteins (dArmRPs) to specifically bind unstructured regions. We assembled a dArmRP to recognize the unstructured KRAS4B-HVR and developed it into a high-affinity binder by directed evolution. The resulting dArmRP recognizes the 14 C-terminal residues of unprocessed KRAS4B, thereby blocking the farnesyltransferase-binding epitope. This steric shielding disrupts KRAS4B post-translational modification and thereby significantly reduces its plasma membrane localization, while demonstrating complete selectivity over KRAS4A, NRAS, and HRAS. This work establishes the shielding of intrinsically disordered regions as a precise biochemical strategy to control protein function and provides an isoform-specific tool to dissect KRAS biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/712636v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@791ac4org.highwire.dtl.DTLVardef@cc4c91org.highwire.dtl.DTLVardef@b6c920org.highwire.dtl.DTLVardef@4e8a9c_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical representation of how the unstructured KRAS4B-HVR is occupied by a dArmRP, making it inaccessible for the FTase.

Targeted protein degradation (TPD) is an emerging therapeutic modality for numerous diseases. PROteolysis-TArgeting Chimeras (PROTACs) represent a potentially generalizable strategy to achieve TPD. A PROTAC is composed of a ligand for a protein of interest, a linker and a ligand for E3 ligase. As such, PROTACs can bring the E3 ligase into the close proximity of a protein target leading to polyubiquitination followed by target protein degradation. While the human genome encodes over 600 E3 ligases, only a handful of them have been harnessed for developing PROTACs. In order to expand the repertoire of E3 ligases for PROTAC development, we developed clickable photoaffinity probes based on clinically used drugs and metabolites to identify potential E3 ligases as the targets. In this paper, we report the discovery of clofibric acid with a molecular weight of only 214 Daltons as a ligand for synoviolin (SYVN1). We demonstrate its utility by developing clofibric acid-based BRD4 PROTACs. The linker length and architecture play a critical role in target degradation efficiency. The clofibric acid-derived BRD4 PROTACs achieve selective BRD4 degradation in a SYVN1-dependent manner. Our findings establish clofibric acid as a robust addition to the TPD toolbox, offering a novel E3 ligase recruitment strategy for the development of next-generation degraders. TOC Graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=90 SRC="FIGDIR/small/701774v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@776850org.highwire.dtl.DTLVardef@1616bd0org.highwire.dtl.DTLVardef@ed3e03org.highwire.dtl.DTLVardef@18271e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Benzoxaboroles offer unusual reactivity and protein recognition for the development of small molecule drugs. Despite this potential, they are uncommon in drug discovery or in large fragment screening libraries. We synthesized a small series of structurally related benzoxaboroles containing a diazirine/alkyne tag to enable in-cell photoaffinity labeling (PAL) experiments. A subset of this library was found to have high selectivity for eukaryotic translation initiation factor 4E (eIF4E). The benzoxaborole-eIF4E interaction was found to be stereoselective in nature and competitive with the 7-methylguanosine cap of mRNA. Site of labeling experiments revealed that the benzoxaborole fragment interacts with the cap binding pocket of eIF4E. In silico modeling of the modified protein suggests that H-bonding interactions between the main chain of Trp102 and the side chain of Asn155 to the amide carbonyl and anionic boronate of the benzoxaborole, respectively, drive affinity for this challenging to drug pocket.

Enzymes used on industrial scale are routinely engineered for best performance. However, exhaustive mutagenesis campaigns using the twenty canonical proteinogenic amino acids rapidly reach an evolutionary ceiling, where gains in activity compromise other critical properties such as thermal endurance. Although non-canonical amino acids (ncAA) expand the chemical space, most are costly for use on an industrial scale and significantly perturb structure. Here, we demonstrate that the evolutionary ceiling of highly optimized polyethylene terephthalate (PET) hydrolases (PETases) can be broken with azatryptophans that (i) differ minimally from their canonical tryptophan, (ii) are genetically encoded, and (iii) are produced in high yield by enzymatic biosynthesis from inexpensive precursors. The first genetic encoding systems are described for 4-azatryptophan, 5-azatryptophan, and 6-azatryptophan, achieving single, site-selective isosteric CH [->] N substitutions that enhancing the catalytic activity while preserving thermal stability. The fluorescence of 6AW provides a uniquely sensitive reporter of side-chain solvent exposure, which is critical for PETase activity and shown to vary between five different PETases. Furthermore, Azatryptophan-bearing enzymes are inexpensive to produce. To benchmark PETase activity, a rapid fluorescence-based kinetic assay, PETra, is introduced, which delivers consistency and reproducibility by using a soluble substrate yet correlates strongly with the hydrolysis of solid PET.

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

We discovered that palmatine (PAL), a well-known natural product, was inducing the fluorogenic fixation of live cells upon visible light irradiation. This ultrafast phenomenon proceeded under high spatiotemporal control down to single cells (SC), with persistence of well-preserved fixed-labeled cells. Cell "optofixing" was mediated by PAL interaction with nuclear and mitochondrial DNA, yielding reactive oxygen species (ROS) mainly singlet oxygen (1O2), lipid peroxidation (LPO) and LPO-derived fixing aldehydes. We found that other DNA dyes including conventional trackers were also capable of optofixing cells, furnishing a consistent methodology (fluorophore-mediated optofixation, FLUMO) across the visible spectrum. Our results pave the way for the functional ablation and labeling of target cell populations using small fluorophores, with applications in SC, organoid and whole organism biology.

Polyketide biosynthesis relies on the conformational adaptability of type II polyketide synthases and accessory enzymes, which direct chain folding and regiospecific cyclization. The aromatase/cyclase TcmN from Streptomyces glaucescensis catalyzes the first two ring closures of tetracenomycin C. Still, the molecular basis by which conformational dynamics regulate substrate binding and product release remains unresolved. Understanding how conformational transitions control ligand recognition and prevent aggregation is crucial for deciphering the molecular bases of polyketide biosynthesis and for guiding engineering strategies to synthesize novel natural products. Here, we investigated how ligand interactions modulate the conformational equilibrium of TcmN and the mechanistic consequences for catalysis. Using NMR spectroscopy (STD, CSP, relaxation dispersion), calorimetry, molecular docking, and microsecond-scale molecular dynamics simulations, we mapped the conformational ensembles of apo TcmN and its complexes with naringenin (a substrate/product analogue) and intermediate 12 (INT12). Apo TcmN samples both open and closed conformations. Naringenin preferentially stabilizes the closed state, a conformation thought to protect hydrophobic residues from solvent exposure. In contrast, INT12 shifts the equilibrium toward the open state, characterized by an expanded cavity that permits substrate entry, product release, and accommodation of extended intermediates. Hydrogen-bond analysis highlighted conserved catalytic residues (R82, E34, Q110, T133) as key anchors for productive poses. These results establish that TcmN functions through a ligand-gated breathing mechanism, in which successive intermediates selectively tune the cavity volume and shape, balancing catalytic efficiency with protection against aggregation. Conformational adaptability emerges as a central determinant of aromatase/cyclase function, providing molecular insights relevant for polyketide biosynthetic engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/708631v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@5646aorg.highwire.dtl.DTLVardef@39016org.highwire.dtl.DTLVardef@1e8c285org.highwire.dtl.DTLVardef@3aba20_HPS_FORMAT_FIGEXP M_FIG C_FIG

Histamine is a key signaling molecule in pathophysiology that can exhibit significant regulatory roles in diverse health and disease status. Besides the well-studied noncovalent interactions between histamine and its receptors, protein histaminylation is a recently discovered mode of action, through which histamine regulates cellular signaling pathways in a covalent-interaction manner. Histaminylation is an emerging protein post-translational modification, where an isopeptide bond is formed between the histamine primary amine and {gamma}-carboxyl group of glutamine through a transamidation reaction catalyzed by transglutaminase 2 (TGM2). However, due to the lack of efficient pan-specific antibodies targeting histaminylated glutamine, the histaminylation proteome in cells remains poorly explored. Here, we report the design and development of a novel N{tau}-propargylated histamine probe as well as its successful application in chemical proteomic profiling of the histaminylation proteome in cancer cells. Notably, new TGM2-catalyzed epigenetic marks on core histones, e.g., H2AXQ84 and Q104 histaminylation, have been identified from cancer cells and verified in this study.

The enzymatic depolymerization of polyethylene terephthalate (PET) presents a sustainable route for plastic circularity, but its industrial viability is disadvantaged by the need for thermostable enzymes that remain active under mild, energy-efficient conditions. While the Polyester Hydrolase Leipzig 7 (PHL7) rapidly degrades amorphous PET near its melting point, its poor protein expression, inactivation issues at temperatures above 60{degrees}C and slow depolymerization activity below 60{degrees}C limit its practical application. Here, we employ inverse folding models ProteinMPNN and LigandMPNN, informed by structural and evolutionary information, to redesign the sequence of PHL7, aiming to improve protein expression, thermal stability and activity. From 36 designed variants, we identified two (termed D5 and D11) with significantly enhanced PET depolymerization rates at lower temperatures, where enzymatic performance is typically limited. Remarkably, design D5 at 50{degrees}C achieved the same product yield as PHL7 at 70{degrees}C in 24 h PET microparticle degradation assays, with a shifted product profile favoring mono-(2-hydroxyethyl) terephthalate (MHET) over terephthalic acid (TPA). Molecular dynamics simulations revealed that the active redesigns exhibit enhanced local flexibility in key active site regions at 50{degrees}C, providing a mechanistic understanding of their low-temperature catalysis. This work demonstrates that computational sequence redesign can optimize biocatalysts for lower production costs and milder operational conditions. Furthermore, the D5 variant enables a potential route to resynthesize virgin PET via MHET polycondensation, offering an efficient circular economy pathway.

Steroid-based fluorescent-quencher probes now enable real-time, residue-level mapping of previously inaccessible cholesterol-binding sites on G-protein-coupled receptors. We designed Tide Quencher 1 (TQ1) conjugated steroids that target two distinct peripheral sites on the M1 muscarinic receptor. One near the extracellular N-terminus and another adjacent to the intracellular C-terminus. Using pregnanolone glutamate as a versatile scaffold, we synthesised a library of probes varying in C-3 linker length ({gamma}-aminobutyric acid vs. L-glutamic acid) and C-3/C-5 stereochemistry (3/3{beta}/5/5{beta}). Fluorescence-quenching assays with CFP-tagged receptors revealed that TQ1 probes consistently outperformed Dabcyl, delivering up to 40 % quenching within minutes and sub-micromolar EC50 values. The most potent N-terminal probe (35-PRG-Glu-TQ1 (5)) achieved 300 nM potency, while the best C-terminal probe (35{beta}-PRG-Glu-TQ1 (3)) reached 1 {micro}M potency with rapid association. Molecular docking and MD simulations identified key residues (K20, Q24, W405 at the N-site; K57, Y62, W150 at the C-site) mediating binding, a prediction confirmed by alanine-scan mutagenesis that markedly reduced quenching at the N-terminus and only modestly affected the C-terminus. Competition experiments with non-quenching analogues further validated probe specificity. Crucially, the pregnane core proved essential; alternative steroid backbones failed to generate robust quenching. This fluorescence-quenching platform overcomes the limitations of traditional radioligand assays, providing kinetic insight, high-throughput compatibility, and the ability to dissect lipid-GPCR interactions in native membranes. The approach is readily extensible to other GPCR families, opening new avenues for structure-guided drug discovery targeting allosteric cholesterol sites.

Viroimmunotherapy leverages oncolytic viruses to induce antitumor immunity and is increasingly explored for solid tumors. Their activity can be enhanced by arming them with immunostimulatory payloads, but most approaches rely on protein-based transgenes that are constrained by viral genome packaging limits. Here, we establish a replication-competent Delta-24-RGD-based platform for localized production of immunotherapeutic RNA aptamers at the tumor site. RNA aptamers provide compact, highly specific ligands that can, in principle, target diverse immune receptors. As a model, we engineered a Delta-24-RGD derivative encoding circular 4-1BB targeting aptamers and show that infected tumor cells sustain aptamer transcription and release, which is associated with a pro-inflammatory remodeling of the tumor microenvironment and measurable antitumor activity in different mouse models with a comparable effect to that achieved with a 4-1BBL-expressing adenovirus used as a benchmark. Overall, this work delivers a proof of concept that replication-competent adenoviruses can serve as in situ factories for extracellularly active RNA aptamers, supporting their development as flexible platforms for localized non-coding cancer immunotherapy.

Protein-protein interactions governed by conformationally heterogeneous domains remain difficult to drug because ligand-competent states are often absent from single static structures. Here, we present AtlasNMR, a statistical framework that transforms multi-model NMR ensembles into screening-ready conformational hypotheses for small molecule discovery. Using the neuronal nitric oxide synthase (nNOS) PDZ domain that engages the adaptor protein CAPON (NOS1AP) as a model system, AtlasNMR identified two representative conformational states capturing the dominant and minor populations of the NMR ensemble. Ensemble-based virtual screening followed by consensus ranking yielded MC-3, a small molecule modulator that disrupts the NOS1-NOS1AP interaction in live cells and directly engages the nNOS PDZ domain. MC-3 produced convergent neuroprotective effects in disease-relevant neuronal models by reducing amyloid-{beta}-induced cytotoxicity, suppressing NMDA-driven nitrosative stress, and attenuating pathological tau phosphorylation, while exhibiting a balanced early lead-like ADME and safety profile. Together, this work establishes a generalizable strategy for exploiting NMR ensemble heterogeneity to enable small molecule discovery against dynamic protein-protein interfaces.

Cell-free gene expression in micro-compartments constitutes a chassis for biotechnology and synthetic biology. Protein synthesis from low concentrations of DNA, a single copy per compartment, is essential for in vitro evolution of biomolecules and synthetic cells. However, insufficient yield of protein synthesized from typically sub-picomolar DNA results in undetectable signals or inadequate activity of desired protein functions. Here we identify and largely mitigate yield-limiting bottlenecks of reconstituted in vitro transcription and translation (IVTT) at low DNA input. Systematic comparison of commercial reconstituted IVTT kits revealed that gene expression starts becoming limited by mRNA scarcity around 20-200 pM DNA input. We further uncovered that the standard ribosome concentration is excessive at low-DNA input and shortens the lifetime of translation. These findings led to a simple optimization recipe that combines supplementation with a highly active T7 RNA polymerase and a reduction in ribosome concentration, which synergistically amplified gene expression by [~]10-fold across diverse fluorescent proteins and enzymes. This low-DNA-optimized formulation in picoliter droplets achieved [~]94 nM protein expression from a single copy of DNA ([~]0.12 pM). The user-friendly boosted IVTT protocol paves the way for straightforward functional screening and in vitro reconstitution of cellular functions in DNA-scarce environments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/707295v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@89a051org.highwire.dtl.DTLVardef@17c2e52org.highwire.dtl.DTLVardef@1c52800org.highwire.dtl.DTLVardef@c543fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Capturing RNA dynamics in living cells would provide critical insights into transcriptional control and cellular adaptation, but remains technically formidable -- particularly at single-base precision. Here, we introduce a DNA tetrahedron based three-dimensional catalytic hairpin assembly (3D@CHA) nanoplatform that couples target recognition with catalytic activation in a spatially organized framework. Three cascaded hairpins (H-AN, H1, and H2) then enable localized and efficient signal amplification. Without external carriers or transfection, the platform exhibits robust biocompatibility, distinguishing highly homologous insulin I (Ins1) and insulin II (Ins2) mRNAs in living cells and tracking their redistribution and intercellular transfer during metabolic changes. Introducing a single-base mismatch site into H1 and coupling it with a Forster resonance energy transfer (FRET) readout yielded a KRAS-3D@CHA probe capable of detecting KRASG12D mutations at the RNA level with single-base resolution. This platform establishes a programmable framework for precise RNA imaging and mutation discrimination, opening new avenues for RNA-level diagnostics and precision oncology.

Genetic code expansion (GCE) enables the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins but is constrained by reliance on exogenously supplied chiral ncAAs. Achieving intracellular ncAA biosynthesis would enable more scalable and cost-effective GCE. Here, we report the continuous hypermutation and evolution of amino acid synthases that produce high levels of ncAAs inside yeast, thus supporting GCE from simple ncAA precursors. We encoded an engineered tyrosine synthase (TmTyrS) on an error-prone orthogonal DNA replication system (OrthoRep) and selected variants based on ncAA biosynthesis from readily available phenol analogs and intracellular L-serine. Our selection employed orthogonal ncAA-specific aminoacyl-tRNA synthetases (aaRSs) as biosensors whereby target ncAA production leads to aminoacylation of an amber suppressor tRNA and the translation of a selectable reporter containing an amber stop codon. Our evolution successfully yielded TmTyrS variants that efficiently produced 3-iodo-, 3-bromo-, 3-chloro-, and 3-methyl-L-tyrosine, enabling amber codon-specified ncAA-dependent translation, in some cases at levels comparable to sense codon-specified natural amino acid translation. This work reduces barriers for expressing proteins containing substituted tyrosines. Moreover, because aaRSs can themselves be evolved (including with OrthoRep) for a flexible range of ncAA specificities, these results establish an end-to-end framework for evolving ncAA biosynthetic enzymes in vivo. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/711232v1_ufig1.gif" ALT="Figure 1"> View larger version (10K): org.highwire.dtl.DTLVardef@6858dorg.highwire.dtl.DTLVardef@2a08b7org.highwire.dtl.DTLVardef@1a24122org.highwire.dtl.DTLVardef@16239a9_HPS_FORMAT_FIGEXP M_FIG C_FIG We describe an OrthoRep-driven platform for evolving noncanonical amino acid (ncAA) synthases. Hypermutation of ncAA synthase genes enables evolution of ncAA biosynthesis from simple precursors, while intracellular ncAA production is linked to fluorescence via an orthogonal aaRS/tRNA system, allowing FACS enrichment of improved variants through iterative cycles.