Chemical Communications

With the increasing interest in RNA-based therapies, there is a pressing need to incorporate new chemistries into more complex RNA molecules. These modifications can protect RNA from degradation, improve its pharmacokinetics, and enhance its targeting properties. Here we describe the enzymatic synthesis of chemically modified RNA derivatives using a mutant T7 RNA polymerase to incorporate 23 different base modifications alongside stabilizing ribose modifications, such as 2'-fluoro and 2'-deoxy groups. To investigate the impact on transcription efficiency and fidelity, we employed a pool of 38 template sequences and analyzed the transcripts by next-generation sequencing of the cDNA. Results demonstrated that all modifications were successfully incorporated into RNA, with transcription efficiency influenced by three main factors: type of modification, base modified, and the sequence context. Misincorporation levels during transcription and reverse transcription into cDNA were generally low (<1%) but included noticeable exceptions for some nucleobase-modification combinations. As a robust proof-of-concept we demonstrated the selection of Histidine-U modified aptamer, relying on multiple rounds of transcription and amplification, binding Influenza hemagglutinin protein with low nanomolar KD. We anticipate that this work will significantly contribute to the design and production of chemically modified RNAs with novel functionalities, advancing applications in biomedicine and synthetic biology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/720138v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@184d010org.highwire.dtl.DTLVardef@77fa67org.highwire.dtl.DTLVardef@d89e2eorg.highwire.dtl.DTLVardef@178ebc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Serotonin is a key neurotransmitter, and aptamer-based tools using the 44 nt Apt44 have been successfully developed for its in vitro and in vivo detection. Nevertheless, the structural basis of recognition by this aptamer remains unclear. Here we report high resolution NMR structures of Apt38, a 6-nt truncated variant in the third loop of Apt44, in free and serotonin-bound states. Both structures reveal a two layered antiparallel chair type G quadruplex core with three edgewise loops and a terminal duplex, forming a G quadruplex duplex hybrid structure. Serotonin binds at the G quadruplex duplex junction, stabilized by stacking, electrostatic attraction, hydrogen bonding, and hydrophobic contacts. Apt38 is preorganized for binding, whereas the longer third loop of Apt44 introduces conformational dynamics into the G quadruplex scaffold, which enables a pronounced binding triggered conformational switch in PBS buffer, explaining its sensing mechanism. Our work reveals the recognition and sensing mechanism of the serotonin aptamer and provides a framework for aptamer design in serotonin biosensing.

The binding of fluorescent dyes to nucleic acids and their fluorogenic properties are indispensable tools for nucleic acid detection, quantification, and imaging, yet the molecular structures of several widely used commercial dyes have remained unknown. Here, we de novo determined the molecular structures of RiboGreen and OliGreen and confirmed the previously proposed structure of PicoGreen using high-field NMR spectroscopy. All three dyes were identified as unsymmetric cyanine dyes, where a benzoxazole/benzothiazole moiety is linked to a 4-quinoline by a monomethine bridge. Complete 1H and 13C resonance assignments enabled us to expand the existing chemical shift reference set for this important class of dyes. Photophysical characterization with standardized single- and double-stranded DNA and RNA targets indicated that all dyes performed similarly upon binding despite being marketed towards different nucleic acid types. NMR spectroscopy and long-timescale molecular dynamics simulations showed that RiboGreen interacts with double-stranded DNA predominantly by two binding modes, electrostatic interactions with the phosphodiester backbone and {pi}-{pi} stacking with the ultimate and penultimate base pairs of the DNA molecule. These results establish the molecular structures of three widely used commercial dyes and provide a structural and mechanistic framework for understanding the fluorogenic properties of this class of dyes. HighlightsO_LIDetermination of the molecular structures of nucleic acid dyes RiboGreen, OliGreen, and PicoGreen C_LIO_LINMR spectroscopic characterization of all three dyes. C_LIO_LINMR and MD data indicate binding to be dominated by electrostatic and {pi}-{pi} stacking interactions C_LI

Biocatalytic metal-containing clusters are nanometer-sized chemical reactors that enable enzymes to perform chemistry far beyond what would be possible with amino acids alone 1,2. These clusters display a remarkable variability3 and elucidating their exact mechanisms is often difficult to determine, requiring input from a multitude of techniques ranging from spectroscopic to structural and theoretical methods. Thus, for unknown clusters, it is essential to accumulate, collate, and interpret as much information as possible from every relevant technique available. Here we report the discovery and in-depth, interdisciplinary characterization of an entirely novel, [Cu-4Fe-4S] cluster in the protein acetol dehydrogenase (AceDH). AceDH, isolated directly from Methylacidiphilum fumariolicum SolV cells, catalyzed the oxidation of acetol to methylglyoxal, proving its role in the 2-propanol/acetone metabolism of methanotrophs4-6. Structural, spectroscopic and electrochemical analyses reveal the [Cu-4Fe-4S] cluster has a unique three-dimensional- and electronic structure involving electronic coupling between the copper and one of the iron atoms, likely contributing to its high, +275 mV, redox potential. The binding site for the novel cluster is composed of two protein subunits and involves a novel motif. These findings expand the known repertoire of biological metal cofactors and provide insight into how heterometallic clusters are adapted for biological processes.

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.

Inorganic polyphosphate (polyP) is a ubiquitous phosphate biopolymer involved in diverse cellular processes. Despite its significance, selective detection of polyP remains challenging because of its simple and highly charged structure. Here, we report a near-infrared (NIR) fluorogenic turn-on chemosensor for selective polyP detection and imaging, SiX-DPA-Zn. The probe combines a silicon-xanthene (SiX) fluorophore with a zinc(II)-coordinated 2,2'-dipicolylamine (DPA-Zn2+) recognition unit and shows more than 100-fold selectivity for inorganic polyP over ADP and ATP. SiX-DPA-Zn enables quantitative detection of polyP at micromolar concentrations in microplate assays and stains a broad range of polyP species, starting from tripolyphosphate, in polyacrylamide gels. In HEK293 cells expressing Escherichia coli polyphosphate kinase 1, the probe visualizes intracellular polyP and enables quantitative analysis of polyP levels in relation to nuclear proteins for example fibrillarin and nucleolin. Stimulated emission depletion (STED) microscopy further revealed subdiffraction-sized polyP granules within polyP aggregates. SiX-DPA-Zn is the first near-infrared (NIR) fluorogenic chemosensor for polyP that is compatible with multiple detection platforms, including microplate assays, polyacrylamide gel staining, confocal and super-resolution STED microscopy.

Curcumin-functionalized gold nanoclusters are promising platforms for catalysis and drug delivery, yet the molecular determinants of their stability, morphology, and solvent response remain unclear. Here, microsecond all-atom molecular dynamics simulations are employed to investigate a 2 nm gold nanoparticle noncovalently coated with different curcumin forms, including neutral enol and trans-keto tautomers, the deprotonated enolate, and their mixtures in water-ethanol and water-methanol solvents. Layer-resolved analyses of radius of gyration, density profiles, and surface coverage reveal that neutral enol and trans forms generate compact assemblies with near-complete surface coverage, whereas enolate-rich systems adopt more expanded conformations with solvent-exposed molecules. Mixed systems preserve these intrinsic packing characteristics while improving overall coverage. Solvent substitution from ethanol to methanol reduces {pi}-{pi} stacking, strengthens Au-curcumin interactions, and increases surface coverage, yielding more compact nanostructures. Free energy and potential of mean force calculations indicate that deprotonated curcumin most effectively screens Au-Au interactions and stabilizes dispersed nanoparticles, while neutral tautomers provide moderate stabilization. Curcumin also enhances the loading of anticancer drug doxorubicin (DOX) onto Au nanoparticles, improving biocompatibility. Enolate(An)-containing systems produce extended structures with weaker membrane interactions, whereas neutral curcumin complexes form compact, positively charged assemblies that strongly bind to negatively charged cancer cell membranes. These findings clarify how tautomeric state and solvent environment cooperatively govern interfacial organization and colloidal stability, establish design guidelines for curcumin-based gold nanocarriers in catalysis, sensing, and drug delivery applications.

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.

L-DOPA is a key therapeutic agent for Parkinsons disease, with growing demand due to global population aging. Here we report that heme-dependent tyrosine hydroxylase (TyrH) can utilize an ascorbate/O2 system--as an alternative to H2O2--to synthesize L-DOPA with markedly enhanced operational stability. While exogenous H2O2 rapidly inactivates TyrH within minutes, sodium ascorbate (NaAsc) enables sustained catalysis for up to 24 h, surpassing the H2O2-driven yield after only 30 min. UV-vis spectroscopy confirms that H2O2 readily degrades the heme center, whereas the heme remains intact in the presence of NaAsc. QM/MM simulations reveal that in situ generated H2O2 leads to the active species of Compound I for tyrosine hydroxylation. Through systematic optimization, we establish efficient reaction conditions (40 {micro}M TyrH, 1 mM L-Tyr, 100 mM NaAsc, pH 8.5, 40 {degrees}C), achieving >95% conversion of L-Tyr to L-DOPA within 2 h. This work not only provides a robust and sustainable biocatalytic route for L-DOPA production but also highlights the broader applicability of the ascorbate/O2 pathway in heme-enzyme catalysis.

We report here a method for the chemical synthesis of Fmocprotected -hydroxyglyine (-OH-Gly) dipeptides. Our method features operational simplicity and is compatible with protecting groups for peptide synthesis. Utility is then demonstrated through substitution at the -OH-Gly position to yield myriad non-natural amino acid-containing dipeptide fragments.

G-quadruplex (GQ) structures within the HIV-1 long terminal repeat (LTR) regulate viral transcription and represent promising antiviral targets; however, detailed mechanistic understanding of their ligand recognition at the molecular level remains limited and has largely been investigated under dilute conditions despite the crowded and compartmentalized nature of intracellular environment. Here, we investigate the interaction of the cationic porphyrin TMPyP4 with the HIV-1 LTR-III GQ under dilute conditions and inside protein-rich phase-separated condensates that mimic intracellular biocondensates. Steady-state and time-resolved fluorescence measurements reveal a dual binding behavior that is not discernible from absorption spectroscopy. A high-affinity guanine-rich binding mode leads to efficient fluorescence quenching through electron transfer from ground-state guanine to excited TMPyP4, whereas a weaker non-guanine binding mode gives rise to enhanced and long-lived emission. Nucleotide-specific control experiments validate the origin of these distinct binding environments. Molecular docking and molecular dynamics simulations further support preferential binding of TMPyP4 at the terminal G-quartet together with a secondary binding mode near the quadruplex-duplex junction. Importantly, both TMPyP4 and LTR-III GQ preferentially partition into the condensates, where the hybrid GQ structure, dual binding behavior, and associated excited-state signatures remain preserved despite the crowded and viscous environment. Although a slight reduction in binding affinity is observed inside the condensates, the overall binding mechanism remains largely preserved due to compensatory effects arising from the condensate microenvironment. Overall, this work demonstrates that ligand recognition of viral GQ remains preserved within protein condensates and establishes fluorescence spectroscopy as a sensitive approach for resolving hidden binding heterogeneity in GQ-ligand interactions.

c-Myc is a transcription factor that drives tumorigenesis in many cancers. It is notoriously difficult to directly target c-Myc, mainly due to its lack of well-defined druggable pockets. O-linked {beta}-N-acetylglucosamine modification (O-GlcNAcylation) is a post-translational modification (PTM) playing an important role in regulating c-Myc functions in cancer. However, previous studies have primarily relied on global perturbations to investigate c-Myc O-GlcNAcylation, making it difficult to determine its direct functional consequences due to concurrent cellular effects. Here, we report a bifunctional O-GlcNAcylation TArgeting Chimera (OGTAC) molecule, which can induce the proximity of c-Myc and O-GlcNAc transferase (OGT) in living cells, thereby enhancing the O-GlcNAcylation of c-Myc. The c-Myc-targeting OGTAC exhibits anti-proliferation effect against cancer cells. Mapping of c-Myc occupancy on genome indicates that OGTAC rewires c-Myc transcriptional activity and reprograms expression of the downstream oncogene MALAT1, in an O-GlcNAcylation-dependent manner. Overall, OGTAC presents a novel chemically induced proximity (CIP)-based tool to target and rewire c-Myc activity in cancer. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/722559v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1c640org.highwire.dtl.DTLVardef@2eb70corg.highwire.dtl.DTLVardef@f38970org.highwire.dtl.DTLVardef@c421c8_HPS_FORMAT_FIGEXP M_FIG C_FIG

The growing number of approved nucleic acid therapeutics illustrates the potential to treat diseases by targeting their genetic blueprints in vivo. The 10-23 DNAzyme is capable of cleaving a wide range of target RNA with high selectivity. However, its poor performance in vivo restricts its therapeutic application as gene silencing agent. Studies on ribozymes have shown that the crowded environment in cells and associated effects can impact ribozyme folding and thermostability, resulting in a change in activity. This opens up the question whether DNAzymes are also affected by molecular crowding. Here, we investigate the functional and structural influence of molecular crowding conditions on the 10-23 DNAzyme. The stability and activity of a PrP-specific 10-23 DNAzyme were examined in presence of PEG, dextran, and osmolytes. Our results indicate that osmolytes decrease DNAzyme activity in a concentration-dependent manner, while certain PEG and dextran concentrations promote activity. To rationalize our observations, we studied the cosolutes effect on physicochemical solution properties and the structure of the DNAzyme:RNA complex using FCS and SAXS. The data reveal that enhanced activity is observed under conditions where a combination of physiochemical properties matches an optimum that seems to be dependent on the metal ion cofactor. Structural influence under such conditions is indicated less. We propose that a certain degree of molecular crowding is required to favor a state, which allows for higher catalytic turnover. In addition, we show that the requirement for magnesium and manganese as a cofactor remains unchanged under the conditions applied. Our work contributes to a better understanding of how the cellular environment affects DNAzyme structure and function.

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.

Fluorescent dyes are critical to visualizing nucleic acids in many applications. SYTOX Orange and SYTOX Green are cyanine dyes, used in dead cell staining and increasingly in single-molecule assays to probe DNA supercoiling and processing. However, their structures and effects on DNA mechanics are not or only partially known. We determine the structure of SYTOX Orange to be (E)-2-((2-(4 ((diethyl(methyl)ammonio)methyl)phenyl)-6-methoxy-1-methylquinolin-4(1H)-ylidene)methyl)-4-methyloxazolo[4,5-b]pyridin-4-ium, identical to SYBR Gold except for an aza-benzoxazol core that is fundamentally different from other dyes in the SYTOX and SYBR families. We report SYTOX Green to be (Z)-2-(bis(3-(trimethylammonio)propyl)amino)-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-1-phenylquinolin-1-ium, similar to PicoGreen. Using magnetic tweezers, we characterize the effect of SYTOX Orange and SYTOX Green on DNA mechanics. They lengthen and unwind DNA consistent with intercalation and the DNA unwinding angles per dye are 21.1(1) degree and 20.5(1) degree for SYTOX Orange and Green, respectively. Both dyes leave the DNA bending persistence length and plectoneme size almost unaltered (<10% change up to 1 uM), which is advantageous in assays probing DNA supercoiling. Their photophysical properties reveal close agreement between single-molecule manipulation and optical absorbance and fluorescence spectroscopy. Our comprehensive set of complementary measurements relates mechanical and optical properties to the molecular structures and provides recommendations for their use in applications.

This study presents the development and spectroscopic characterization of an erlotinib-functionalized gold nanoparticle (erlotinib:AuNP) nanosystem designed for targeted delivery to metastatic non-small cell lung cancer H1299 cells. Initial MTS assays demonstrated that free erlotinib induced a concentration-dependent reduction in cell viability, while 0.1 {micro}M erlotinib exhibited negligible cytotoxicity and was therefore selected for nanosystem fabrication. AuNPs alone showed minimal toxicity toward H1299 cells over the investigated concentration range. Following conjugation of erlotinib with AuNPs, the resulting nanosystems reduced cell viability to approximately 60%, indicating enhanced biological activity of the drug after nanoparticle-assisted delivery. Fluorescence microscopy confirmed the intracellular internalization of the nanosystems in H1299 cells, with nanoparticle aggregates predominantly localized in the perinuclear and perimitochondrial regions. Three-dimensional Raman spectroscopy (3D RS) mapping further verified the intracellular localization of the conjugates through characteristic Raman signatures of erlotinib:AuNPs. Importantly, 3D RS enabled detection of nanosystems at concentrations below the sensitivity limit of fluorescence imaging, demonstrating superior analytical performance for intracellular nanosystem tracking. Atomic force microscopy-infrared (AFM-IR) spectroscopy coupled with principal component analysis (PCA) demonstrated substantial biochemical modifications induced by the erlotinib:AuNP nanosystems, including enhanced lipid-related spectral features and significant alterations in protein secondary structure, particularly the increased contribution of unordered and antiparallel {beta}-turn conformations. The obtained results demonstrate that combining plasmonic nanocarriers with advanced vibrational spectroscopy enables highly sensitive monitoring of intracellular drug delivery and nanosystem-induced biochemical responses in cancer cells.

Misfolded proteins are tightly associated with various neurodegenerative diseases, and removing these misfolded proteins is one of the actively pursued approaches for seeking therapeutics for these diseases. In this study, we demonstrated that molecularly produced light (molecular light) from ADLumin-5, a self-photosensitizing chemiluminescence compound, could induce photo-oxidation and photodegradation of misfolded proteins, including beta-amyloid, tau, alpha-synucleins, and TDP-43 proteins in vitro. We validated the oxidation and degradation via LC-MS, MADLI-MS, and western blotting. Using beta-amyloid as a showcase, we demonstrated that, upon photo-oxidation and photodegradation, the toxicities of this misfolded protein were significantly reduced. To investigate the therapeutic effects of ADLumin-5 in vivo, we used the 5xFAD mouse model for longitudinal treatment for 4 months. In vivo molecular imaging results indicated that ADLumin-5 could reduce the accumulation of beta-amyloid proteins. Our study presents a novel approach to seek therapeutics for neurodegenerative disease via molecular light-induced degradation of misfolded proteins. In addition, because ADLumin-5 is dual-functional--enabling both photodegradation and in vivo imaging of misfolded protein changes--it can be considered a photo-theranostic agent for neurodegenerative diseases, representing a novel approach to drug discovery for neurodegenerative diseases.

Proteins in solution adsorb to the corona of nanoparticles such as single-walled carbon nanotubes (SWCNTs), but these interactions are difficult to predict and analyze due to ambiguities in the structure of the latter. In this work, we employ ss(GT)15-DNA wrapped SWCNTs, a commonly used fluorescent sensor construct, to examine protein adsorption by quantifying binding dissociation constants and characterizing the corresponding photophysical effects. A library of 20 proteins are used to evaluate adsorption-induced changes in photoluminescence (PL) intensity ({Delta}I/I0) and emission wavelength upon solution phase binding. We find that 15 proteins produce monotonic dose-response behavior well described using a single-site Langmuir model. Alternatively, five proteins exhibited more complex, non-monotonic behavior consistent with a two-step binding model representing protein-protein interactions coupled to adsorption. The study reveals that metalloproteins, which comprised 12 of the 20 proteins in the library, induced greater PL quenching compared with metal-free proteins for this system, with maximum binding-associated quenching ({Delta}I/I0) of 94% for metalloproteins versus 20% for metal-free proteins. For metalloproteins, we introduce a proximity-based quenching framework in which protein size provides a coarse proxy for cofactor-SWCNT separation, offering a mechanistic interpretation of the observed quenching variation across proteins. Together, these results establish the use of metal coordination sites, such as those in metalloproteins, to assist the transduction of certain nanoparticle fluorescent sensors, helping with sensor probe design and interpretation in biological environments.

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

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