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.

In this work we present antibody-metal conjugate as a new subclass of antibody-drug conjugates (ADC) for the chemodynamic therapy of cancer based on the rapid generation of reactive oxygen species (ROS) upon copper reduction. We used conventional therapeutic antibody trastuzumab and DOTA-NHS ester for the design and initial proof-of-concept. Thus, trastuzumab-DOTA-copper conjugate (TDCC) was synthesized. We demonstrate that TDCC retains specific binding to HER2-positive cancer cells with approximately native immunoreactivity and achieves stable copper incorporation with an average drug-to-antibody ratio of up to [~]8. In the presence of physiological reducing agents such as N-acetylcysteine or cysteine, TDCC generates substantial reactive oxygen species (ROS), leading to pronounced cytotoxicity and long-term suppression of clonogenic survival in HER2-positive SK-BR-3 and BT-474 cells. Notably, HER2-negative MDA-MB-231 cells and non-malignant HS5 fibroblasts remain largely unaffected, confirming target-dependent activity. The conjugate remains stable under storage conditions for up to 30 days, and the DOTA linker itself does not interfere with copper-mediated redox chemistry. Our findings identify TDCC as a novel class of targeted oxidative stress inducers that exploit the vulnerability of HER2-positive tumors to copper-mediated cytotoxicity. This strategy not only preserves the specificity of antibody-based delivery but also introduces a distinct mechanism of action capable of bypassing conventional resistance pathways, warranting further preclinical development. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/721915v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@7ed6bdorg.highwire.dtl.DTLVardef@1442b2aorg.highwire.dtl.DTLVardef@6dff28org.highwire.dtl.DTLVardef@18aba16_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Phosphorodiamidate morpholino oligomers (PMOs) are approved exon-skipping antisense therapeutics for Duchenne muscular dystrophy (DMD), but their clinical utility is limited by poor uptake in muscle tissue, necessitating frequent high-dose administration. Cell-penetrating peptides (CPPs) can enhance intracellular delivery of PMOs, yet conventional arginine-rich CPPs often cause dose-limiting toxicity, including renal damage, which hinders their clinical translation. To address this challenge, we developed a high-throughput, charge-based chromatographic enrichment platform capable of screening over 15,000 synthetic peptides, including sequences with noncanonical (abiotic) amino acids. This approach enabled de novo discovery of arginine-depleted CPPs with improved delivery profiles. Four lead candidates demonstrated efficient nuclear PMO delivery with ~10-fold lower in vitro toxicity compared to standard CPPs such as penetratin. The top-performing peptide, CXP1, showed robust splice-switching activity and favorable tolerability in both cellular and animal models. In dystrophic mdx mice, CXP1-PMO conjugates achieved greater exon skipping compared to PMOs conjugated to R6G at equivalent doses. Tissue levels of CXP1-PMO correlated with exon-skipping efficacy, establishing a clear pharmacokinetic-pharmacodynamic relationship. These findings highlight a mechanistically novel and translationally relevant discovery strategy, demonstrating the potential of high-throughput platforms to generate more effective CPP-based delivery vehicles for antisense therapeutics in DMD and related neuromuscular disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=44 SRC="FIGDIR/small/730741v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@84b7f2org.highwire.dtl.DTLVardef@1487850org.highwire.dtl.DTLVardef@d2e263org.highwire.dtl.DTLVardef@10ce371_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

Virus-like particles represent an emerging and promising vaccine platform. However, these particles are inherently mechanically soft and have limited control over particle surface architecture, thereby constraining their immunological control. Herein, we report the rational design of bioinspired virus-like porous silica (VLPSi) nanoparticles (NPs) with tunable and mechanically rigid spike architectures that function dually as antigen delivery carriers and immune adjuvants. Using ovalbumin (OVA) as a model antigen, we systematically elucidate the spiky structure-function relationship in antigen delivery and immune response. VLPSi NPs exhibit good biocompatibility, sustained antigen release, and markedly enhanced cellular uptake and endosomal escape compared with soft spike and spherical counterparts. Mechanistic investigations combining molecular dynamics simulations and proteomic analyses reveal that rigid spike architectures reduce the energetic barrier for cellular internalization and concurrently activate dual pathways involving endosomal Toll like receptors and calcium signaling. Consequently, VLPSi with long spikes elicit significantly enhanced humoral and cellular immune responses, outperforming the particles with shorter spikes, spherical shape as well as clinically used alum adjuvant. To demonstrate translational potential, bioinspired antibacterial vaccines were produced by loading Staphylococcus aureus surface protein rEsxB. The VLPSi-based vaccine elicited robust protective immunity to achieve complete (100%) survival following lethal challenge without detectable adverse effects, whereas traditional Alum-adjuvanted formulation conferred only minimal protection, with a survival rate of 10%. Collectively, this work establishes VLPSi with tunable spikes as a mechanistically controlled platform for next generation vaccines. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC="FIGDIR/small/720861v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1f6c13eorg.highwire.dtl.DTLVardef@1090d07org.highwire.dtl.DTLVardef@1364926org.highwire.dtl.DTLVardef@fc68ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that enables cellular adaptation to environmental, nutritional and metabolic cues. Upon ligand binding, AHR translocates to the nucleus, heterodimerizes with the AHR nuclear translocator (ARNT) and regulates gene expression. Current approaches to measure AHR activity rely on transcriptional readouts, which vary depending on cell type and ligand. Here, we introduce two complementary protein-protein interaction-based assays that detect AHR activation by monitoring AHR-ARNT complex formation. Split-luciferase (NanoBiT) and bimolecular fluorescence complementation (BiFC) detect AHR activation independently of transcriptional output, capturing agonist- and antagonist-dependent AHR modulation across multiple ligands and cellular contexts. NanoBiT enables rapid, real-time analysis of AHR dimerization, whereas BiFC supports imaging of AHR interactions at subcellular resolution. The assays capture further attributes of AHR signaling, including dissociation from chaperones or HIF-1-mediated competition for ARNT, and enable detection of AHR activation in biological samples. Hence, both assays provide versatile tools to study AHR signaling.

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.

Solid-phase peptide synthesis (SPPS) remains the dominant technique for peptide production. However, its reliance on hazardous organic solvents such as N, N-dimethylformamide (DMF) and dichloromethane (DCM) results in an adverse environmental burden. One potential approach is replacing these organic solvents with water to reduce the hazardous solvent consumption and improve the environmental footprint of peptide production. This has led to the emergence of aqueous solid-phase peptide synthesis (ASPPS) approaches. Although successful, these approaches require specialized hydrophilic resins or modified building blocks, limiting their industrial applicability and scalability. Moreover, conventional hydrophobic polystyrene supports, remain the most widely used solid supports in industrial SPPS due to their high loading capacity, mechanical robustness, and low cost. These resins are generally considered incompatible with aqueous conditions. Here, we demonstrate that industrially relevant 2-chlorotrityl chloride (CTC) polystyrene resin can support efficient peptide coupling under fully aqueous conditions by integrating a precipitate-free 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC{middle dot}HCl) and Oxyma activation system with a synergistic thermal-acoustic strategy. We posit that heating combined with ultrasonic irradiation likely promotes transient relaxation of the polystyrene matrix and enhances water penetration. This facilitates the diffusion of activated amino acid esters onto the hydrophobic resin required for coupling. The robustness of this aqueous methodology was validated through the synthesis of nine structurally diverse peptide sequences, including aromatic hydrogel-forming peptides, opioid peptides derived from enkephalins, toxin-inspired sequences, and a lipid-interacting fragment of -synuclein. Analytical characterization by HPLC and MALDI-TOF mass spectrometry confirmed successful peptide assembly with high crude purity. We anticipate that this thermal-acoustic aqueous SPPS strategy provides a scalable and accessible pathway toward sustainable peptide manufacturing on classical hydrophobic supports with aqueous chemistry.

RNA therapeutic modalities such as antisense oligonucleotides (ASOs) have emerged as promising tools to target previously "undruggable" targets. Despite their great promise as precision therapeutic agents, their clinical adoption remains limited due to production costs, sequence-length restrictions, limited structural heterogeneity, and the generation of environmentally hazardous waste during synthesis. Biocatalytic synthesis strategies provide a sustainable alternative; however, their reliance on specialized enzymes and precursors often limits sequence diversity and scalability. To address these limitations, we report the design and biocatalytic synthesis of a novel circular ASO: Nanostructured Form-switching Antisense Oligonucleotide (NaFASO) for targeting Japanese Encephalitis Virus (JEV) infection-associated host long non-coding RNA (lncRNA) JINR1 (JEV-induced non-coding RNA1) in SH-SY5Y cells. The novel modular architecture in NaFASO has been designed to have a metastable stem that separates the functional antisense domain from the splint-padlock circularizing region, ensuring both structural integrity and efficient target engagement. The serum- and nuclease-stable NaFASOs achieved knockdown of the lncRNA JINR1 during JEV infection, resulting in a reduction in JEV replication and neuronal cell death. NaFASO-mediated JINR1 depletion also resulted in downregulation of the JEV replication-associated gene GRP78. Together, these findings establish NaFASO as a first-of-its-kind structure-switching circular ASO platform for combating JEV infection, combining stability, efficacy, and environmental sustainability. Beyond the JEV, the generalizability of this design suggests broad applicability for targeting diverse RNA species implicated in genetic disorders, viral infections, and cancer, thus highlighting a promising paradigm for developing next-generation transformational nucleic acid therapeutics.

Calcineurin (Cn) is a protein phosphatase that initiates T-cell activation by dephosphorylating the transcription factor NFAT, driving its nuclear translocation and the transcription of immune-related genes. While clinical immunosuppressants like Cyclosporine A (CsA) potently inhibit Cn, they completely block its catalytic site, leading to non-specific inhibition and severe off-target toxicity. Selectively targeting the specific protein-protein interaction (PPI) between Cn and NFAT presents a safer therapeutic strategy. We previously identified the C16orf74 (C16) peptide as a high-affinity Cn-NFAT PPI inhibitor; however, its utility in cellular systems is restricted by poor membrane permeability. In this study, we evaluated cell-penetrating peptide (CPP) conjugates of C16 with an N-terminus transactivator of transcription (TAT) and polyarginine (R11) to enable efficient intracellular delivery. Structural modeling, fluorescence polarization displacement, and pull-down assays confirmed that the CPP-C16 conjugates retain the ability to compete with an NFAT-derived peptide and bind Cn. Fluorescence microscopy demonstrated efficient intracellular entry of TAT-C16 and R11-C16 in mammalian cells, and effective inhibition of NFAT nuclear translocation and attenuation of downstream NFAT-dependent transcriptional activity of the IL-2 gene in human T cells at concentrations of 10 {micro}M or lower. Crucially, unlike CsA, the CPP-C16 peptides exhibited minimal cytotoxicity even at high concentrations of up to 50 {micro}M, establishing a potential safe therapeutic window. These findings establish CPP-C16 conjugates as effective, cell-permeable, and non-toxic inhibitors of the Cn-NFAT signaling axis, providing the basis for the development of PPI-directed immunosuppressants.

Oligonucleotide therapeutics such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) enable sequence-specific gene silencing but rely on endogenous cellular machinery and often require extensive chemical modification for stability and efficacy. DNAzymes offer a mechanistically distinct alternative through intrinsic catalytic RNA cleavage; however, their therapeutic translation has been limited by nuclease susceptibility, structural constraints, and synthetic challenges. Here, we report the development of Circular Active Nano DNAzyme (CANDe), an enzymatically synthesized circular DNAzyme platform designed to enhance stability without backbone modification. The therapeutic potential of CANDe constructs was investigated against Japanese Encephalitis Virus (JEV) infection associated host long-noncoding RNA JINR1 (LINC01518). CANDe constructs were generated via splint-assisted ligation and incorporate modular elements, including catalytic cores (8-17 or 10-23), target-binding arms, and structural stems. Circularization conferred marked resistance to exonuclease-mediated degradation compared to linear DNA, maintaining structural integrity under nuclease-rich conditions.,CANDe targeting the lncRNA JINR1 achieved effective JINR1 knockdown in SHSY-5Y with and without JEV infection. This was accompanied by reduced expression JEV RNA and titers. In line with this, CANDe constructs attenuated of virus-induced cytotoxicity and apoptosis. Among the constructs, 10-23-based CANDe targeting the JINR1-1 site exhibited the strongest overall activity. These findings establish CANDe as a modular, modification-free DNAzyme platform that combines catalytic efficiency with enhanced stability, enabling effective host-directed antiviral intervention. This approach highlights topological engineering as a viable alternative to chemical modification for advancing DNAzyme-based therapeutics.

RNA-targeting small molecules are emerging as promising therapeutic modalities, but their development requires methods that define binding sites and evaluate RNA target selectivity. Existing approaches for detecting ligand-RNA interactions have provided powerful foundations, yet many rely on direct crosslinking or covalent-capture chemistries whose performance depends on ligand-specific probe design, warhead compatibility, and local reaction geometry. Here, we report Singlet Oxygen footprinting on RNA in a Ligand-Directed manner for Mutational Profiling (SOLiD-MaP), a photoproximity labelling platform for small molecule-RNA interaction analysis. Using the Mango-II aptamer and thiazole orange derivatives as a model system, we establish aniline as an efficient nucleophile for singlet oxygen-mediated RNA labelling and demonstrate target-selective labelling driven by ligand-localized photosensitization. We further show that labelling selectivity can be tuned by chemically constraining the singlet oxygen diffusion with a quencher. Finally, we develop a pairwise reverse transcription stop assay and a mutational profiling with next-generation sequencing readouts to infer ligand-proximal regions and unambiguously map binding sites. SOLiD-MaP provides a new, orthogonal strategy for studying small molecule-RNA recognition and should support RNA-focused mechanism-of-action studies.

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.

Selective chemical modification of RNA is essential for RNA functionalization, probing RNA structure-function relationships and developing RNA-targeted therapeutics. Existing chemical strategies often rely on guanine accessibility or multiple helper DNA strands, restricting their generality and biological applicability. Inspired by DNA-guided DMAP catalysis and small-molecule binding-induced crosslinking, we report a small molecule-directed, DMAP-catalyzed, proximity-driven strategy for site-selective RNA functionalization. By appending a catalytic DMAP moiety to RNA-binding ligands, 2'-OH groups are selectively acylated in the presence of azide-bearing acyl donors, enabling subsequent installation of bioorthogonal handles. This approach was validated across diverse RNAs, including Pepper and Clivia RNA aptamers, G-quadruplex Broccoli RNA, and endogenous FMN riboswitch RNA. For a 400-nt Pepper-7SK fusion, selective modification of the Pepper motif was achieved with minimal perturbation to the nucleus localization function of 7SK RNA. Optimized PEG-pentafluorophenyl (PFP) acyl donors provided enhanced reactivity and low background. The method operates catalytically, decouples ligand recognition from the labeling moiety, and enables selective enrichment of target RNAs, offering a versatile platform for RNA functionalization, ligand profiling, and potentially live-cell applications.

Enzyme cascades enable complex biochemical transformations, but their optimization is resource-intensive, requiring navigation through high-dimensional parameter spaces encompassing reaction conditions, enzyme ratios, and buffer composition. Here we introduce CascadeMAP, an autonomous microfluidic platform for closed-loop optimization of enzyme cascades, integrating high-throughput microfluidics with Bayesian optimization and multi-agent AI system. We demonstrate the platform across two cascades: (i) a glycerol detection pathway monitored by fluorescence and (ii) a 1,2,3-trichloropropane degradation pathway monitored by label-free Raman spectroscopy providing orthogonal detection modalities. Bayesian optimization identified optimal conditions three times faster than Design of Experiments. Multi-agent AI system automated hypothesis generation, processing 11 GB of experimental data, pattern recognition, and insight synthesis. Operating without human intervention for 7 days, CascadeMAP processed [~]220,000 reactions across [~]7,400 different conditions. This capability establishes a generalizable framework for the autonomous optimization of enzyme cascades and metabolic pathways and accelerates the development of biocatalytic and synthetic biological systems.

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.

Protein glycosylation and phosphorylation are fundamental post-translational modifications (PTMs) that coordinate cellular signaling. While receptor tyrosine kinases (RTKs) like the epidermal growth factor receptor (EGFR) are heavily glycosylated, the systems-level crosstalk between extracellular sialylation and intracellular phosphorylation dynamics remains poorly understood. We employed an integrated TMT18-labeled multi-omics pipeline to simultaneously profile the global proteome, phosphoproteome, and N-glycoproteome of A498 cells. Using enzymatic in-situ remodeling with sialidase, we investigated the signaling response to EGF stimulation and the synergistic effects of desialylation with the tyrosine kinase inhibitor (TKI) gefitinib. Our analysis revealed that cell surface desialylation significantly attenuates EGF-induced signaling, specifically suppressing over 200 phosphosites within the MAPK cascade and actin cytoskeleton organization modules. Comparative profiling demonstrated that sialidase treatment exerts a distinct regulatory program that is non-redundant with canonical TKI inhibition. Stoichiometric analysis confirmed that the depletion of sialylated N-glycoforms at specific EGFR residues (N413, N444) directly correlates with reduced phosphorylation at key activation sites (Y1197). Finally, an integrated glyco-phospho network analysis identified CD44, MET, and integrin signaling hubs as central nodes regulated by the sialylation. This study establishes cell surface sialylation as a critical rheostat for EGFR-mediated signaling flux. By bridging the gap between the extracellular glycoprotein and intracellular kinase networks, we identify glycan remodeling as a potent strategy to sensitize RTK-driven malignancies to therapy. Our findings provide a robust data foundation for developing glycoconjugate-targeted interventions in ccRCC and beyond.

Phosphate is central to modern bioenergetics and to all theories for the origin of life. How phosphate entered metabolism is unknown, though microbial physiology and geochemical environments can provide important clues. Some bacteria obtain electrons and energy from phosphite (HPO32-), a reduced form of phosphate (HPO42-), that naturally occurs in serpentinizing (H2-producing) hydrothermal systems. Here we show that the insoluble, solid-state catalyst native palladium, which is naturally deposited in serpentinizing hydrothermal systems, catalyzes the oxidation of phosphite to phosphate and H2 in water at 25-100 {degrees}C in a highly exergonic reaction. Palladium awaruite (PdxNi3Fe), a common form of Pd0 in serpentinizing vents, also catalyzes phosphite-dependent phosphorylation. Phosphite oxidation over Pd0 generates a reactive but so far unidentified chemical intermediate, possibly metaphosphate, [PO3]-, that readily phosphorylates hydroxyl moieties in glycerol, ribose, glucose, serine and cytidine at 25-100 {degrees}C in 2-72 h. The same conditions also generate (i) phosphoanhydride bonds in pyrophosphate, polyphosphates and ADP, (ii) the phosphoramidate bond in phosphocreatine, (iii) and the acyl phosphate bond in acetyl phosphate, which is obtained overnight at 25 {degrees}C with 8% yield. The reactions proceed without sulfur, excluding thioester or metal sulfide intermediates. Phosphite-dependent phosphorylations under serpentinizing hydrothermal vent conditions are facile. They identify a natural, geochemical source of prebiotic phosphorylation and a novel source of metabolic energy at origins. The central role of phosphate in bioenergetics, metabolism, and nucleic acids could reflect metal-catalyzed, redox chemistry of phosphorus in the environment where metabolism (and life) arose.

Cells depend on the spatial organization of proteins, RNA, and DNA into discrete subcellular compartments. Previous methods have largely centered on measuring spatial organization based on only one of these biomolecular classes at a time. Here, we demonstrate that POCA photocatalytic proximity labeling can serve as a unified photosensitizer-based platform for profiling the proximal proteomes of protein, RNA, and DNA targets within a single experimental framework. We show that POCA can harness standard immunofluorescence or in situ hybridization workflows to specifically target organic fluorophore photosensitizers to intracellular targets for proximity labeling in fixed cells. POCA-targeted proximity labeling requires minimal cellular input and does not require genetic engineering. Additionally, POCA photosensitizers are selected to also be fluorescent, enabling direct confirmation of on-target localization by imaging prior to proteomic analysis. To demonstrate broad utility, we apply POCA across multiple molecular targets spanning protein, RNA, and genomic DNA, including components of the nuclear pore complex, nucleolus, nuclear speckles, telomeres, and pericentromeric heterochromatin. By anchoring proximity labeling to both a protein and an RNA within the same nuclear compartment, we resolve shared and distinct proximal proteomes from orthogonal molecular perspectives.