Angewandte Chemie

Interactions between proteins and glycosphingolipids (GSLs) regulate various cellular processes and altered GSL metabolism contributes to numerous diseases. The diverse glycan headgroups and ceramide backbones of GSLs shape membrane organisation, fluidity, curvature, and tension. As protein recognition frequently depends on both glycan specificity and the organisation of GSLs within the membrane, these interactions remain challenging to characterise in vitro. Here, we introduce FLiPA (Fluorescent Liposome Plate Assay), a versatile method that utilises fluorescent agarose-embedded giant liposomes for the quantitative analysis of protein-GSL interactions. By enabling systematic control of membrane and buffer composition, FLiPA provides an accessible and robust platform for dissecting the molecular determinants of protein-GSL interactions, including the roles of cholesterol, membrane order, protein oligomerisation and ionic strength.

Norepinephrine (NE) is a key neurotransmitter and hormone involved in diverse physiological and pathological processes. Beyond its canonical non-covalent signaling through adrenergic receptors, NE also induces protein post-translational modifications (PTMs), representing an emerging regulatory mechanism. Two major forms of NE-derived PTMs have been identified: non-enzymatic norepinephrinylation (NEylation) of cysteine residues mediated by NE quinone and transglutaminase 2 (TG2)-catalyzed NEylation of glutamine residues. However, the biochemical basis and pathophysiological roles of NEylation remain poorly understood due to limited detection tools. Here, we report a bioorthogonal reaction for selective labeling and enrichment of the NEylation proteome in cell lines and tissues, which is based on acid-catalyzed dehydration and 1,6-addition to thiol probes. This strategy enables fluorescence imaging and chemical proteomic profiling, revealing NEylation as a widespread PTM that affects enzymatic activities of modified proteins, including protein tyrosine-protein phosphatase non-receptor type 11 (PTPN11). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/731782v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19dce3aorg.highwire.dtl.DTLVardef@14e7f30org.highwire.dtl.DTLVardef@80354aorg.highwire.dtl.DTLVardef@12ac6f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.

The spontaneous adsorption of biomolecules onto nanoparticle surfaces has been extensively characterized at the protein level, but the metabolite corona remains poorly defined while being physicochemically and biologically distinctive. Herein, we report the first class-level mapping of metabolite corona composition across 25 chemically modified carbon nanotubes in human plasma using untargeted liquid chromatography-mass spectrometry. Complementary analytical conditions detected approximately 9,000 metabolite features, of which over 5,000 yielded valid corona-versus-plasma enrichment measurements. Machine learning classifiers extended metabolite class annotations from 10-45% to the full detected set, enabling systematic analysis of class-level enrichment patterns. We find that polymer wrapping dominates corona composition, with DNA wrapping selectively enriching nonpolar lipids and PEG wrapping favoring polar metabolites. Within each polymer background, covalent quantum well defects further modulate class-level enrichment in a structure- and chemistry-dependent manner. Carboxyl aryl defects broadly enhance amphiphilic lipid recruitment, while trifluoro aryl defects suppress single-chain amphiphilic species but attenuate depletion of double-chain phospholipids. Our findings demonstrate that engineered nanotubes can serve as chemically tunable, selective scaffolds for metabolite enrichment, potentially enhancing the capability of nanotube-based platforms to recruit and detect structurally diverse, low-abundance small molecules in complex biofluids.

Nanoscale lipid bilayer mimetics are powerful tools for research on lipid bilayer, membrane proteins or for drug delivery. Established nanoscale bilayer systems that are stabilized by short peptides or polymers produce a broad size distribution and are difficult to customize. Here we introduce a DNA nanotechnology-based lipid bilayer mimetic, in which we covalently conjugated established nanodisc-forming amphiphilic peptides to oligonucleotides. These peptide-DNA conjugates were then hybridized with a circular single-stranded scaffold to form stiff, circular PDC minicircles with 14 peptide modifications at the inner rim of the torus. Lipid reconstitution yielded defined nanodisc with a tightly controlled circumference and component stoichiometry. Molecular dynamics simulations further validated the structural stability and reveal an asymmetric migration of the DNA to one rim of the bilayer. To mimic membrane protein insertion, we co-reconstituted a transmembrane peptide coupled to a bulky quantum dot. In future applications, the size and peptide arrangement can easily be modified in these DNA-templated PDC nanodiscs.

Conformationally responsive DNA nanoswitches have previously been developed and validated for a variety of biosensing applications including detection of DNA, microRNA, and viral RNA/DNA. Here we develop new methodology for enhancing the sensitivity of DNA-based sensing by recycling a fixed number of targets for repeated reuse. We achieved target-dependent enzymatic ligation of looped nanoswitches and showed that subsequent removal of target does not affect the ligated loop. Through cyclic annealing, ligation, and target removal, we can linearly control signal amplification up to hundreds of cycles. This method adds an important new capability for low abundance targets without the need for target amplification.

Huntingtons disease is a fatal neurodegenerative disorder caused by expansion of a cytosine-adenosine-guanine repeat in exon 1 of the Huntingtin (HTT) gene, resulting in a polyglutamine-expanded HTT protein. Although the genetic cause of Huntingtons is well defined, the molecular functions of HTT and the mechanisms linking polyglutamine expansion to neurodegeneration remain incompletely understood. Huntingtin Associated Protein 40 kDa (HAP40) is a key HTT interaction partner that forms a stable complex with HTT and is increasingly recognized as an important player in the HTT structure-function paradigm. However, investigation of HAP40 biology has been limited by a lack of tools capable of directly targeting endogenous protein. Here, we report the discovery and characterization of a panel of nanomolar-affinity macrocyclic peptides targeting HAP40 identified using Random nonstandard Peptide Integrated discovery platform. We characterized macrocycle binding in vitro using surface plasmon resonance, fluorescence polarization, and hydrogen-deuterium exchange mass spectrometry, revealing selective, high-affinity engagement of distinct epitopes on HAP40. We further demonstrate that these macrocycles engage endogenous HAP40 in cellular lysates, enable selective isolation of HAP40-containing protein complexes using macrocycle precipitations and insights into interaction partners of distinct HTT and HAP40 proteoforms. Together, these macrocycles establish a new toolkit for investigating HTT-HAP40 biology and provide a framework for dissecting HAP40-specific functions relevant to Huntingtons disease pathogenesis.

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.

Activating KRAS mutations drive millions of cancers diagnosed worldwide,1 yet for decades this oncoprotein was deemed "undruggable", reflecting the challenge of discovering molecules capable of perturbing its complex biological functions, and of translating these discoveries into effective cancer therapeutics.2 Recent advances propelled by innovative screening have identified diverse modalities that bind at or near the switch-II pocket (SII-P) of RAS proteins, including molecular glues,3 macrocyclic peptides,4 fragment-derived small molecules,5 and approved therapies that covalently target KRASG12C.6,7 Unfortunately, resistance to approved therapies has emerged,8,9 highlighting the need for molecules that engage new or underexploited binding sites on RAS oncoproteins with mechanisms complementary to established SII-P inhibitors.10,11 Here we show that mirror-image mRNA display12 enabled the discovery of all-D macrocyclic peptide ligands targeting a cryptic RAS back pocket (CRB-P).13 These ligands engage KRAS(OFF) and KRAS(ON) with equal affinity, exploit a single-residue difference among isoforms to bind KRAS selectively, and successfully inhibit oncogenic signaling in KRAS-mutant cells through a mechanism distinct from SII-P binders. Mirror-image screening directly afforded nanomolar peptide ligands stable toward cellular proteolysis and delivered probes targeting distinct epitopes not accessible by homochiral peptide-display methods. Together, these findings establish the CRB-P as a specifically druggable and mechanistically differentiated site on KRAS with potential for combination with emerging RAS-targeting therapies and substantiate mirror-image mRNA display as a strategy for discovering stable all-D macrocyclic peptides targeting previously inaccessible epitopes on challenging targets.

Oncogene amplification is a key driver of tumorigenesis and a perpetuator of genomic instability. Oncogene amplification accelerates cancer cell proliferation and evolution, contributing substantially to the enhancement of adaptation mechanisms, such as treatment resistance, which pose a significant therapeutic challenge. However, previous studies have shown oncogene amplification to be a critical vulnerability, rendering cancer cells, but not normal cells, susceptible to targeted, CRISPR-Cas9 nickase - mediated DNA damage and cell death in vitro. Here, we demonstrate the initial framework for the translation of this potential therapeutic approach utilizing Cas9D10A - mRNA and functionalized lipid nanoparticles for the targeted delivery, and suppression of disseminated MYCN-amplified neuroblastoma in vivo.

Atomically precise gold nanoclusters (AuNCs) are ultra-small particles composed of ten to hundreds gold atoms and exhibit unique photophysical properties. Significant progress has been made in tuning and extending their luminescence in the near-infrared window through the design of AuNC assemblies. Herein, we report a straightforward method for synthesizing highly pure, programmable DNA tetrahedra functionalized with a controlled number of AuNCs (from one up to four AuNCs). Using ligand exchange chemistry, AuNCs bearing a single grafted ssDNA onto them were produced. These constructs then served as building blocks for synthesizing tetrahedra through DNA hybridization. Products obtained at each stage of the synthesis were thoroughly characterized using a range of complementary technics. Notably, mass spectrometry in native mode provided novel insights into the accurate composition and stoichiometry of these architectures. This study paves the way for the synthesis and the characterization of a variety of new three-dimensional, DNA-guided AuNC assemblies that may serve as powerful theranostics and biophotonic tools.

The multivalent display of surface glycoprotein hemagglutinin (HA) on Influenza A viruses (IAVs) enhances the overall binding avidity to sialylated glycans on host cell surfaces. While precomplexing HA trimers with antibodies increases multivalency and avidity, this method does not replicate the virions geometry and limits insights into the multivalent binding process. Here, we use perfectly controllable icosahedral protein nanoparticles to examine the multivalent HA receptor-binding properties. We compare three HA presentation systems with varying degrees of multivalency: single HA trimers, antibody-precomplexed HA trimers, and HA trimers on nanoparticles. Our results indicate that increasing HA valency enhances binding avidity across various glycan surfaces, including erythrocytes, cells, and lipid bilayers with varying glycan densities, while maintaining receptor specificity. By combining functional and non-functional HA trimers during nanoparticle formation, we create statistical mixtures of nanoparticles with varying valencies. At high receptor densities, nanoparticles with few functional trimers still bind strongly, whereas at low receptor densities, a patch of five HA trimers appears necessary for binding. As a key finding, we observe that such a statistical mixture of nanoparticles with functional and nonfunctional HAs binds to glycan surfaces in a stronger density-dependent manner than fully functional particles. We also observe differences in binding modes that correlate with the number of functional trimers, the glycan structure (linear vs branched), and the densities achievable with these glycans. Overall, our findings demonstrate that the presentation of multivalent HA plays an enormous role in the response to glycan receptor type and density, with implications for the future design of virus monitoring, viral inhibitors, and targeting vectors.

DNA-programmed assembly of plasmonic nanostructures provides a powerful route to couple molecular recognition with optical signal generation. Here, we report the sequence-specific assembly of DNA-functionalized gold nanorods using a sesame allergen-derived DNA biomarker as a molecular bridge. Target-induced assembly produces concentration-dependent assembly growth, plasmon coupling, and distinct assembly kinetics that are readily monitored by absorption spectroscopy, enabling label-free detection of the target DNA in the nanomolar concentration range. The assembled nanorods further produce strong surface-enhanced Raman scattering (SERS) signals arising from plasmonic coupling within the assemblies, extending detection to the picomolar regime without the use of Raman reporters. Quantitative analysis reveals that both the extent and rate of assembly formation are governed by target DNA concentration. These results establish a direct relationship between molecular recognition, assembly growth, plasmonic coupling, and spectroscopic response, highlighting DNA-programmed gold nanorod assembly as a versatile platform for investigating hybridization-driven plasmonic self-assembly and nucleic acid detection. Table of Content O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/732610v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@6a8f42org.highwire.dtl.DTLVardef@1e36b9corg.highwire.dtl.DTLVardef@1ade546org.highwire.dtl.DTLVardef@1a787bd_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

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.

Cell-free biosensors leverage in vitro gene expression reactions to detect chemicals. While inexpensive, modular, and distributable, these platforms are constrained by slow readouts at ambient temperatures, precluding practical field operation. In cells, phase separation accelerates biochemical reactions; however, recapitulating these gains in vitro has remained challenging for complex biochemistries. Here, we report the first self-assembling coacervate system that accelerates in vitro transcription. Prepared by simple mixing, coacervation with spermine and polyacrylic acid occurs dynamically in response to NTP consumption and co-localizes DNA templates and RNA polymerase to accelerate transcription, mimicking intracellular phenomena. We exploit this discovery to accelerate the cell-free biosensing of six ligands, demonstrating that coacervation can preserve platform modularity, improve sensitivity, retain lyophilization compatibility, function in field matrices, and reduce ambient-temperature time-to-signal by hours. This work contributes to a growing understanding of phase separation in biology and advances the use of membrane-less organization for real-world applications.

Chemical labeling of nucleic acids is essential to pinpoint the structure, localization, and function of RNA and DNA. Yet, reversible sequence-independent chemistries that can label native RNA and DNA remain poorly developed. Here we describe Reversible Uridine Nitrilium-mediated Addition (RUNA), a reversible covalent chemistry that selectively modifies uridine and thymidine residues via N3 deprotonation and reaction with a nitrilium ion intermediate generated from an aldehyde and an isonitrile. The reaction forms a stable N3 adduct that can be quantitatively reversed by hydrolysis. By using reagents that are either membrane permeable or impermeable, we demonstrate the localization and function of DNA on exosomes. Although exosomes harbor nucleic acids, whether the latter are encapsulated in the exosome lumen or are surface-adhered is unknown. RUNA revealed that exosomes display DNA on their outer surface. The abundance of such surface DNA increases upon DNA-damage accumulation in cancer cells that are treated with a PARP inhibitor. This surface DNA drives exosome uptake by M2-polarized macrophages through scavenger receptors and triggers a shift toward an M1-like pro-inflammatory state. The selective labeling of surface DNA revealed an unexpected mechanism by which exosomes engage innate immune cells. RUNA is a versatile tool to analyze the nucleic acid content and functionality of extracellular vesicles in health and disease. Significance StatementPinpointing the localization of RNA and DNA in cells and organelles is central to deriving insights into their biological functions in health and disease. We describe a new method, RUNA, for labeling nucleic acids that is sequence-independent and reversible. By varying RUNA reagents, we can distinguish between nucleic acids that are located either inside or outside of membrane compartments. Using RUNA, we showed that DNA is associated with the outer surface of exosomes that are secreted by cancer cells. Further, the amount of surface DNA increases when the cancer cells are treated with an anti-cancer drug. This surface DNA promotes the uptake of exosomes by innate immune cells known as macrophages and modulates their inflammatory response.

Efficient extrahepatic delivery of siRNAs remains a major limitation for broadening their therapeutic potential. Using a modular, orthogonal click chemistry platform, we generated 28 siRNA conjugates varying in ligand class, valency, and spatial arrangement. Following systemic administration, fatty acid conjugates - particularly palmitic acid (C16) - outperformed sterol- and phospholipid-based designs in promoting extrahepatic gene silencing, with preferential activity observed in heart and skeletal muscle. Increasing ligand valency through 3',5'-bis-conjugation generally enhanced activity compared to 5-mono conjugation. Nevertheless, bis-C22 conjugates showed increased hepatic activity, suggesting a shift in tissue distribution linked to hydrophobicity. Architectural parameters further modulated outcomes: Branched 5' C16 conjugates, bearing two lipids on one terminus, were markedly less active than their bis counterparts and required short PEG spacers to restore activity. Notably, bis-lipid conjugation strategies that enhanced extrahepatic activity for an siRNA did not translate to an ASO gapmer, underscoring modality-specific constraints. Together, these findings delineate structure-activity relationships and establish bis-fatty-acid conjugation as a robust design principle for achieving extrahepatic RNAi. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/726808v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@287a47org.highwire.dtl.DTLVardef@17407eborg.highwire.dtl.DTLVardef@b40435org.highwire.dtl.DTLVardef@804352_HPS_FORMAT_FIGEXP M_FIG C_FIG

The ability to encode and reliably read nanoscale information is increasingly important for multiplexed biomolecular detection and super-resolution imaging. DNA origami provides a uniquely programmable platform for arranging structural and functional elements with nanometer precision, enabling the creation of identifiable nanoscale patterns. In this context, DNA origami-based barcodes that incorporate gold nanoparticles (AuNPs) to encode either origami geometry or the identity of specific biological targets within defined nanoparticle patterns have been paired with transmission electron microscopy imaging for decoding. However, surface-bond AuNPs may detach during handling, purification, or biological incubation, leading to misidentification or decoding errors in barcode analysis. Here we report a rational design for the controlled encapsulation of AuNPs within DNA origami tubes to enhance nanoparticle retention and structural integrity. We engineered curvature-inducing modifications in a flat rectangular DNA origami scaffold to promote inward folding and confinement of AuNPs. These barcodes can be further functionalized on the outer surface with bioactive aptamers and/or fluorescence dyes, enabling targeted interactions with cells and optical readout. Programable dimerization further expands multiplexing capacity. This design provides a robust framework for structurally stable origami barcodes and advances the development of high-resolution, multiplexed labeling and diagnostic platforms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/725969v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@686c1aorg.highwire.dtl.DTLVardef@1914c4eorg.highwire.dtl.DTLVardef@28ad47org.highwire.dtl.DTLVardef@8847ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Sphingomyelin is the most abundant sphingolipid in mammalian cells and is synthesized by two isoenzymes, sphingomyelin synthase 1 and 2, located in the Golgi and in the plasma membrane. Abnormal sphingomyelin synthesis is associated with infections and diseases such as diabetes and cancer. Measuring cellular sphingomyelin synthase activity fosters our understanding of how these enzymes are involved in pathological processes and can be crucial for the identification of therapeutic compounds modifying sphingomyelin biosynthesis. We have developed a novel fluorometric assay that enables microscopic detection of cellular sphingomyelin synthase activity. We show that sphingomyelin synthases use propargyl choline and -NH2-{omega}-N3-C6-ceramide to generate trifunctional sphingomyelin, a lipid derivative detectable with spatial resolution via Forster resonance energy transfer. By combining this assay with expansion microscopy, a super-resolution imaging technique, we measured the distribution of de novo synthesized trifunctional sphingomyelin in cells at nanoscale resolution, thereby directly demonstrating sphingomyelin biosynthesis at the Golgi and the plasma membrane. By monitoring sphingomyelin biosynthesis and degradation in cells infected with the obligate intracellular pathogen Chlamydia trachomatis, we dissected the complex sphingolipid metabolization of these bacteria with unprecedented resolution. By correlating spatial metabolic information with lipidomics, we provide a powerful tool for investigating cellular sphingomyelin metabolism. TeaserCombining functional lipids, expansion microscopy and FRET helps visualise sphingolipid metabolism at unprecedented resolution.