ABSTRACT
Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.
Subject(s)
Coloring Agents/chemistry , Molecular Probes/chemistry , Nonlinear Optical Microscopy/methods , Optical Imaging/methods , Pyronine/chemistry , Coloring Agents/chemical synthesis , HeLa Cells , Humans , Models, Chemical , Molecular Probes/chemical synthesis , Molecular Structure , Pyronine/analogs & derivatives , Pyronine/chemical synthesis , Spectrum Analysis, Raman/methods , VibrationSubject(s)
Bone Neoplasms , Gold/chemistry , Metal Nanoparticles/chemistry , Osteosarcoma , Photosensitizing Agents , AC133 Antigen/metabolism , Animals , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cell Survival/drug effects , Female , Humans , Male , Mice , Mice, Nude , Molecular Probes/chemistry , Molecular Probes/metabolism , Osteosarcoma/metabolism , Osteosarcoma/pathology , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Photosensitizing Agents/pharmacology , Phototherapy , Theranostic Nanomedicine/methodsABSTRACT
Positron emission tomography (PET)-fluorescence imaging is an emerging field of multimodality imaging seeking to attain synergy between the two techniques. The probes employed in PET-fluorescence imaging incorporate both a fluorophore and radioisotope which enable complementary information to be obtained from both imaging techniques via the administration of a single agent. Fluorine-18 is the most commonly used radioisotope in PET imaging and consequently many novel attempts to radiofluorinate various fluorophores have transpired over the past decade. In this Minireview, the most relevant fluorine-18 labelled PET-fluorescence probes have been classified into four groups as per the implemented fluorophore: 1)â boron-dipyrromethene (BODIPY) dyes, 2)â cyanine dyes, 3)â alternative organic fluorophores and 4)â organometallics, such as quantum dots (QDs) and rhenium complexes. The biological, radiochemical and photophysical properties of each probe have been systematically compared to aid future endeavours in PET-fluorescence chemistry.
Subject(s)
Fluorescence , Fluorine Radioisotopes/chemistry , Molecular Probes/chemistry , Positron-Emission Tomography , Fluorescent Dyes/chemistryABSTRACT
Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron-donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3 orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple centers. New electrophiles and their corresponding alkyne conjugates were profiled directly in cultured cells, achieving thiol saturation in a few minutes at submillimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable the mass spectrometry discovery of >3000 reactive and/or accessible thiols labeled in their native cellular environments in a fraction of the standard analysis time. Surprisingly, only half of the annotated cysteines were identified by both iodoacetamide-desthiobiotin and methylsulfonylbenzothiazole-desthiobiotin in replicate experiments, demonstrating complementary detection by mass spectrometry analysis. These probes offer advantages over existing cysteine alkylation reagents, including accelerated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide analysis and covalent ligand discovery.
Subject(s)
Cysteine/chemistry , Sulfones/chemistry , Alkynes/chemistry , Indicators and Reagents/chemistry , Molecular Probes/chemistry , Oxidation-Reduction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Photoacoustic (PA) imaging as a noninvasive biomedical imaging technology exhibits high spatial resolution and deep tissue penetration for in vivo imaging. In order to fully explore the potential of PA imaging in biomedical applications, new contrast agents with improved PA stability and efficiency are in high demand. Herein, we present a new PA agent based on an oxygen-embedded quinoidal nonacene chromophore that is self-assembled into nanoparticles (Nano(O-Nonacene)-PEG), assisted by polyethylene glycol (PEG). Notably, the photothermal conversion efficiency of Nano(O-Nonacene)-PEG is 1.5 fold that of semiconducting polymer nanoparticles (Nano(PCPDTBT)-PEG) and 2.8 fold that of Au nanorods, owing to the low quantum yield of Nano(O-Nonacene)-PEG. Thereby, Nano(O-Nonacene)-PEG possess a greatly elevated PA signal intensity, compared to Nano(PCPDTBT)-PEG and Au nanorods, which have been widely explored for PA imaging. Due to the high resistance to photo bleaching, Nano(O-Nonacene)-PEG exhibits higher PA signal stability, which may be employed for long-term PA imaging. Moreover, when magnetic Zn0.4Fe2.6O4 nanoparticles are incorporated into Nano(O-Nonacene)-PEG, not only are magnetic resonance signals generated but also the photoacoustic efficacy is greatly enhanced. Therefore, Nano(O-Nonacene)-PEG offers distinct properties: (i) the elevated photoacoustic effect allows for high-resolution photoacoustic imaging, (ii) small size (10 nm in diameter) results in efficient tumor-targeting, and (iii) the facile application of efficient photothermal therapy in vivo. The current work offers the possibility of oxygen-embedded quinoidal acene as a promising PA probe for precision phototheranostics.
Subject(s)
Molecular Imaging , Molecular Probes/chemistry , Nanoparticles/chemistry , Oxygen/chemistry , Photoacoustic Techniques , Phototherapy , Quinones/chemistry , Molecular Structure , Particle Size , Polyethylene Glycols/chemistry , Semiconductors , Surface PropertiesABSTRACT
A strategy for the photoelectrochemical detection of miRNA with ultra-low background noise was developed using tungsten diselenide-cysteine-dopamine (WSe2/Cys/DA) as a nanoprobe coupled with mismatched catalytic hairpin assembly target recycling. A superior detection limit of 3.3 aM toward miRNA-221 was achieved.
Subject(s)
Cysteine/chemistry , Dopamine/chemistry , Electrochemical Techniques/methods , MicroRNAs/analysis , Molecular Probes/chemistry , Nanostructures , Photochemical Processes , Selenium/chemistry , Tungsten/chemistry , Biosensing Techniques , Catalysis , Humans , Limit of Detection , MicroRNAs/blood , Proof of Concept StudyABSTRACT
A straightforward method for the preparation of non-K region fused phosphorus- and nitrogen-containing (PN)-heterocyclic pyrenes has been accomplished. Their structural, photophysical, and electrochemical properties have been fully investigated. The described compounds exhibit intriguing redox properties and show strong photoluminescence with a wide range of tunable emission colors, with λem ranging from blue at 459 nm to red at 622 nm in CH2Cl2 solution.
Subject(s)
Pyrenes/chemistry , Crystallography, X-Ray , Cyclization , Density Functional Theory , Electrochemical Techniques , Fluorescence , Luminescence , Molecular Probes/chemistry , Molecular Structure , Nitrogen/chemistry , Oxidation-Reduction , Phosphorus/chemistry , Pyrenes/chemical synthesis , Quantum Theory , Solvents/chemistryABSTRACT
Multifunctionalized materials are expected to be versatile probes to find specific interactions between a ligand and a target biomaterial. Thus, efficient methods to prepare possible combinations of the functionalities is desired. The concept of dynamic combinatorial chemistry (DCC) is ideal for the generation of any possible combination, as well as screening for target biomaterials. Here, we propose a new molecular design of multitopic probes for ligand discovery in DCC. We synthesized a new Gable Porphyrin, GP1, having prop-2-yne groups as a scaffold to introduce various functional groups. GP1 is a bis(imidazolylporphyrinatozinc) compound connected through a 1,3-phenylene moiety, and it gives macrocycles spontaneously and quantitatively by strong imidazole-to-zinc complementary coordination. Some different types of functional groups were introduced into GP1 in high yields. Formation of heterogeneous macrocycles composed of GP1 derivatives having different types of substituents was accomplished under equilibrium conditions. These results promise that enormous numbers of macrocycles having various functional groups can be provided when the kinds of GP components increase. These features are desirable for DCC, and the present system using GP1 is a potential candidate to provide a dynamic combinatorial library of multitopic probes to discover specific interactions between a ligand and a biomaterial.
Subject(s)
Combinatorial Chemistry Techniques/methods , Drug Discovery , Molecular Probes/chemistry , Dimerization , Isomerism , Ligands , Models, Molecular , Porphyrins/chemical synthesis , Porphyrins/chemistry , Proton Magnetic Resonance Spectroscopy , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Zinc/chemistryABSTRACT
INTRODUCTION: Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose (FDG) presents some limitations for imaging of hepatocellular carcinoma (HCC), the most common primary hepatic malignancy. AREAS COVERED: The authors discuss the accuracy and limitations of FDG for HCC detection. Then, authors examine the recent advances in PET tracers other than FDG for the biological and prognostic characterization of HCC such as 11C-acetate, 11C-choline, and its 18F-labeled derivatives. EXPERT COMMENTARY: FDG PET can be helpful for the identification of the more aggressive and poorly differentiated HCC. 11C-acetate is readily incorporated into intracellular phosphatidylcholine membranes and proved useful for the in vivo biological characterization of the more differentiated and less aggressive HCC. Nevertheless, the short half-life of 11C- radionuclide limits the clinical application of this compound. 11C-choline, another surrogate biomarker of cell membrane biosynthesis, has been demonstrated effective for HCC imaging. The availability of choline derivatives labeled with 18F-radionuclide (i.e. 18F-fluoroethylcholine, 18F-fluorocholine) has overcome the drawbacks due to 11C, thus triggering the clinical applications of choline PET for HCC diagnosis and management. Further research needs to be conducted to better define the alternative or complementary role of these PET probes for the characterization of HCC, with particular regard to the dual-tracer PET-CT modality.
Subject(s)
Carcinoma, Hepatocellular/diagnostic imaging , Liver Neoplasms/diagnostic imaging , Molecular Probes/chemistry , Positron-Emission Tomography , Humans , Molecular ImagingABSTRACT
Carbon dots (CDs) with low biotoxicity, high photostability, and well-controlled size are highly desirable imaging agents for optical bioimaging. However, most of the CDs triggered by ultraviolet/blue light present visible/first near-infrared emissions shorter than 820 nm, impairing their imaging applications in vivo by low penetration depth. Hence, developing novel CD-based materials with second near-infrared (NIR-II) emission located in 1000-1700 nm region is an urgent task. Here, a novel NIR-II-emitting CD-based nanoprobe triggered by 808 nm laser is developed. The designed CDs with 900-1200 nm luminescence possess high quantum yield (QY-0.4%) and high biocompatibility, which have proven to be effective probes for in vivo NIR-II bioimaging. Notably, nearly 65% CDs are excreted from mouse urine within 6 h, demonstrating the rapid renal clearance of CDs. Furthermore, the designed CDs also exhibit high photothermal efficiency (30.6%), making them ideal materials for thermal ablation of cancer. Our findings pave the way of designing a multifunctional CD-based theranostic platform for simultaneously integrating the advanced NIR-II bioimaging and photothermal therapy of cancer.
Subject(s)
Carbon/metabolism , Infrared Rays , Kidney , Optical Imaging/methods , Quantum Dots/metabolism , Theranostic Nanomedicine/methods , Animals , Carbon/chemistry , Carbon/pharmacokinetics , HeLa Cells , Humans , Kidney/diagnostic imaging , Kidney/metabolism , Mice , Molecular Probes/chemistry , Molecular Probes/metabolism , Molecular Probes/pharmacokinetics , Phantoms, Imaging , Phototherapy , Quantum Dots/chemistry , Spectroscopy, Near-InfraredABSTRACT
Flake-shaped nanohybrids based on black phosphorus (BP) have been developed as multifunctional theranostic nanoplatforms for drug delivery, phototherapy and bioimaging. In this work, we report a facile strategy for fabrication of black phosphorus-Au nanoparticle hybrids (BP-AuNPs), which reveal an extraordinary near-infrared (NIR) photothermal transduction efficiency and drug delivery capacity. The applications of the nanocomposites as therapeutic agents for high-performance chemo-photothermal tumor therapy are accomplished in vitro and in vivo. BP-AuNPs also exhibit wonderful surface-enhanced Raman scattering (SERS) activity under NIR laser excitation with a low Raman background, allowing BP-AuNPs to be used as a promising two-dimensional (2D) fingerprint nanoprobe for bio-SERS analysis. The cellular component identification and label-free live-cell bioimaging based on this type of 2D SERS substrate are generally investigated, which open up promising new perspectives in nanomedicine, including diagnosis, imaging and therapy.
Subject(s)
Gold , Metal Nanoparticles , Molecular Probes , Neoplasms, Experimental/diagnostic imaging , Neoplasms, Experimental/drug therapy , Photochemotherapy , Animals , Gold/chemistry , Gold/pharmacology , Metal Nanoparticles/chemistry , Metal Nanoparticles/therapeutic use , Mice , Molecular Probes/chemistry , Molecular Probes/pharmacologyABSTRACT
A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.
Subject(s)
Alkynes/chemistry , Azides/chemistry , Lactose/analogs & derivatives , Molecular Probes/chemistry , beta-Galactosidase/analysis , Ampicillin/pharmacology , Anti-Bacterial Agents/pharmacology , Aspergillus oryzae/enzymology , Aspergillus oryzae/growth & development , Click Chemistry , Copper/chemistry , Cycloaddition Reaction , Enzyme Assays/methods , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/growth & development , Microbial Sensitivity Tests , Molecular Probe Techniques , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , beta-Galactosidase/chemistryABSTRACT
Understanding the functional role of glycosylation-mediated pathogenesis requires deep characterization of glycoproteome, which remains extremely challenging due to the inherently complex nature of glycoproteins. We demonstrate the utility of lectin-magnetic nanoprobe (MNP@lectin) coupled to Orbitrap HCD-CID-MS/MS for complementary glycotope-specific enrichment and site-specific glycosylation analysis of the glycoproteome. By three nanoprobes, MNP@ConA, MNP@AAL, and MNP@SNA, our results revealed the first large-scale glycoproteome of nonsmall cell lung cancer (NSCLC) with 2290 and 2767 nonredundant glycopeptides confidently identified (Byonic score ≥100) in EGFR-TKI-sensitive PC9 and -resistant PC9-IR cells, respectively, especially with more fucosylated and sialylated glycopeptides in PC9-IR cells. The complementary enrichment was demonstrated with only five glycopeptides commonly enriched in three MNP@lectins. Glycotope specificity of 79 and 62% for enrichment was achieved using MNP@AAL and MNP@SNA, respectively. Label-free quantitation revealed predominant fucosylation in PC9-IR cells, suggesting its potential role associated with NSCLC resistance. Moreover, without immunoprecipitation, this multilectin nanoprobe allows the sensitive identification of 51 glycopeptides from 10 of 12 reported sites from onco-protein EGFR. Our results not only demonstrated a sensitive approach to study the vastly under-represented N-glycoprotome but also may pave the way for a glycoproteomic atlas to further explore the site-specific function of glycoproteins associated with drug resistance in NSCLC.
Subject(s)
Carcinoma, Non-Small-Cell Lung/chemistry , Glycopeptides/isolation & purification , Glycoproteins/isolation & purification , Lectins/chemistry , Lung Neoplasms/chemistry , Proteome/isolation & purification , Tandem Mass Spectrometry/methods , Amino Acid Sequence , Carbohydrate Sequence , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Glycoconjugates/chemistry , Glycoconjugates/metabolism , Glycopeptides/classification , Glycopeptides/genetics , Glycopeptides/metabolism , Glycoproteins/classification , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Humans , Lectins/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Magnetite Nanoparticles/chemistry , Molecular Probes/chemistry , Molecular Probes/metabolism , Proteome/classification , Proteome/genetics , Proteome/metabolism , ProteomicsABSTRACT
Studies on the human proteome have engaged diverse techniques; however, none of them represent a predominant approach. Chemical biology has made a major contribution to our understanding of human biology, stimulating the generation of biological hypotheses. Tools such as functional probes have advanced studies on biological mechanisms and helped in elucidating off-target reactivity and potential toxicities of drugs and drug candidates. Here, we accentuate the recent developments in the design and applications of phosph(on)ate-containing probes. Phosphate esters and anhydrides are present in a number of vital cell constituents, and their significance can be reflected by a number of biological processes that involve phosphorus-bearing molecules. We discuss the use of phosph(on)ate-derived probes for (1) the identification of phosphate-requiring enzymes, their substrates, interacting partners; (2) developing screening assays; and (3) their potential as diagnostics. Limitations that as yet need to be overcome and possible measures to be undertaken will also be addressed.
Subject(s)
Molecular Probes/chemistry , Phosphorus/chemistry , Proteins/chemistry , Proteome/chemistry , Animals , Humans , Phosphorus/metabolism , Proteins/metabolism , Proteome/metabolismABSTRACT
Prolonged drug-target occupancy has become increasingly important in lead optimization, and biophysical assays that measure residence time are in high demand. Here we report a practical label-free assay methodology that provides kinetic and affinity measurements suitable for most target classes without long preincubations and over comparatively short sample contact times. The method, referred to as a "chaser" assay, has been applied to three sets of unrelated kinase/inhibitor panels in order to measure the residence times, where correlation with observed efficacy was suspected. A lower throughput chaser assay measured a residence time of 3.6 days ±3.4% (95% CI) and provided single digit pM sensitivity. A higher throughput chaser methodology enabled a maximum capacity of 108 compounds in duplicate/day with an upper residence time limit of 9 h given an assay dissociation time of 34 min.
Subject(s)
Biosensing Techniques/methods , Drug Evaluation, Preclinical/methods , Azo Compounds/chemistry , Biosensing Techniques/instrumentation , Biotin/metabolism , Drug Evaluation, Preclinical/instrumentation , High-Throughput Screening Assays/instrumentation , High-Throughput Screening Assays/methods , Models, Theoretical , Molecular Probes/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinases/chemistry , Protein Kinases/metabolism , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Streptavidin/metabolism , Structure-Activity Relationship , Time FactorsABSTRACT
BACKGROUND: Ag2S has the characteristics of conventional quantum dot such as broad excitation spectrum, narrow emission spectrum, long fluorescence lifetime, strong anti-bleaching ability, and other optical properties. Moreover, since its fluorescence emission is located in the NIR-II region, has stronger penetrating ability for tissue. Ag2S quantum dot has strong absorption during the visible and NIR regions, it has good photothermal and photoacoustic response under certain wavelength excitation. RESULTS: 200 nm aqueous probe Ag2S@DSPE-PEG2000-FA (Ag2S@DP-FA) with good dispersibility and stability was prepared by coating hydrophobic Ag2S with the mixture of folic acid (FA) modified DSPE-PEG2000 (DP) and other polymers, it was found the probe had good fluorescent, photoacoustic and photothermal responses, and a low cell cytotoxicity at 50 µg/mL Ag concentration. Blood biochemical analysis, liver enzyme and tissue histopathological test showed that no significant influence was observed on blood and organs within 15 days after injection of the probe. In vivo and in vitro fluorescence and photoacoustic imaging of the probe further demonstrated that the Ag2S@DP-FA probe had good active targeting ability for tumor. In vivo and in vitro photothermal therapy experiments confirmed that the probe also had good ability of killing tumor by photothermal. CONCLUSIONS: Ag2S@DP-FA was a safe, integrated diagnosis and treatment probe with multi-mode imaging, photothermal therapy and active targeting ability, which had a great application prospect in the early diagnosis and treatment of tumor.
Subject(s)
Molecular Probes , Optical Imaging/methods , Photoacoustic Techniques/methods , Quantum Dots , Silver Compounds , A549 Cells , Animals , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Male , Mice , Mice, Inbred BALB C , Molecular Probes/chemistry , Molecular Probes/toxicity , Phosphatidylethanolamines/chemistry , Phosphatidylethanolamines/toxicity , Phototherapy , Polyethylene Glycols/chemistry , Polyethylene Glycols/toxicity , Quantum Dots/chemistry , Quantum Dots/toxicity , Silver Compounds/chemistry , Silver Compounds/toxicityABSTRACT
Single molecule localization microscopy (SMLM) is a powerful tool for imaging biological targets at the nanoscale. In this report, we present SMLM imaging of telomeres and centromeres using fluorescence in situ hybridization (FISH). The FISH probes were fabricated by decorating CdSSe/ZnS quantum dots (QDs) with telomere or centromere complementary DNA strands. SMLM imaging experiments using commercially available peptide nucleic acid (PNA) probes labeled with organic fluorophores were also conducted to demonstrate the advantages of using QDs FISH probes. Compared with the PNA probes, the QDs probes have the following merits. First, the fluorescence blinking of QDs can be realized in aqueous solution or PBS buffer without thiol, which is a key buffer component for organic fluorophores' blinking. Second, fluorescence blinking of the QDs probe needs only one excitation light (i.e. 405 nm). While fluorescence blinking of the organic fluorophores usually requires two illumination lights, that is, the activation light (i.e. 405 nm) and the imaging light. Third, the high quantum yield, multiple switching times and a good optical stability make the QDs more suitable for long-term imaging. The localization precision achieved in telomeres and centromeres imaging experiments is about 30 nm, which is far beyond the diffraction limit. SMLM has enabled new insights into telomeres or centromeres on the molecular level, and it is even possible to determine the length of telomere and become a potential technique for telomere-related investigation.
Subject(s)
Centromere/metabolism , In Situ Hybridization, Fluorescence , Quantum Dots/chemistry , Semiconductors , Single Molecule Imaging/methods , Telomere/metabolism , Cadmium Compounds/chemistry , HeLa Cells , Humans , Lasers , Molecular Probes/chemistry , Quantum Dots/ultrastructure , Sulfides/chemistry , Zinc Compounds/chemistryABSTRACT
Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.
Subject(s)
E1A-Associated p300 Protein/analysis , Molecular Probes/chemistry , Phosphines/chemistry , E1A-Associated p300 Protein/genetics , E1A-Associated p300 Protein/metabolism , Histones/metabolism , Humans , Lysine/metabolism , Protein Processing, Post-TranslationalABSTRACT
Regardless of the applications: therapeutic vehicle or membrane model to mimic complex biological systems; it is of a great importance to develop simplified, reproducible and rapid model assays allowing for a relevant assessment of the liposomal membrane oxidation and therefore antioxidant activity of selected molecules. Here, we describe a new and high-throughput assay that we called "Vesicle Conjugated Autoxidizable Triene (VesiCAT)". It is based on specific UV absorbance spectral properties of a new phospholipid probe, synthesized with natural conjugated eleostearic acid extracted from Tung oil. The VesiCAT assay has been developed with two different radical generators (2,2'-azobis(2-amidinopropane) dihydrochloride; AAPH and 2,2'-azobis(2,4-dimethylvaleronitrile); AMVN), producing a constant flux of oxidant species, either in membrane or in aqueous phase. This method appears very efficient in assessing the effect of various pure antioxidant molecules in their ability to preserve liposomes from oxidative degradation. In addition, the AAPH- and AMVN-induced oxidations offer the possibility of extracting different but complementary information with respect to the antioxidants efficacy.
Subject(s)
Antioxidants/chemistry , Linolenic Acids/chemistry , Liposomes/chemistry , Molecular Probes/chemistry , Phospholipids/chemistry , High-Throughput Screening Assays , Oxidation-Reduction , Phospholipids/chemical synthesis , Plant Oils/chemistryABSTRACT
The cysteine hydrolase, N-acylethanolamine acid amidase (NAAA) is a promising target for analgesic and anti-inflammatory drugs. Here, we describe the development of two unprecedented NAAA-reactive activity-based probes as research tools for application in the discovery of new inhibitors and for the in-depth characterization of NAAA in its cellular environment.