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1.
Sci Signal ; 14(675)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758061

RESUMO

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from acidic organelles through the activation of two-pore channels (TPCs) to regulate endolysosomal trafficking events. NAADP action is mediated by NAADP-binding protein(s) of unknown identity that confer NAADP sensitivity to TPCs. Here, we used a "clickable" NAADP-based photoprobe to isolate human NAADP-binding proteins and identified Jupiter microtubule-associated homolog 2 (JPT2) as a TPC accessory protein required for endogenous NAADP-evoked Ca2+ signaling. JPT2 was also required for the translocation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus through the endolysosomal system. Thus, JPT2 is a component of the NAADP receptor complex that is essential for TPC-dependent Ca2+ signaling and control of coronaviral entry.


Assuntos
/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , NADP/análogos & derivados , /fisiologia , Marcadores de Afinidade , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Química Click/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transcriptoma , Internalização do Vírus
2.
Carbohydr Polym ; 260: 117812, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33712157

RESUMO

A dual pH-/thermo-responsive hydrogel was designed based on a polyelectrolyte complex of polyacrylic acid (PAA) and norbornene-functionalized chitosan (CsNb), which was synergized with chemical crosslinking using bistetrazine-poly(N-isopropyl acrylamide) (bisTz-PNIPAM). The thermo-responsive polymeric crosslinker, bisTz-PNIPAM, was synthesized via reversible addition-fragmentation transfer polymerization of NIPAM. FTIR, XRD, rheological and morphological analyses demonstrated the successful formation of the polyelectrolyte network. The highly porous structure generated through the in-situ "click" reaction between Tz and Nb resulted in a higher drug loading (29.35 %). The hydrogel (COOH/NH2 mole ratio of 3:1) exhibited limited drug release (8.5 %) of 5-ASA at a pH of 2.2, but it provided an almost complete release (92 %) at pH 7.4 and 37 °C within 48 h due to the pH responsiveness of PAA, hydrogel porosity, and shrinkage behavior of PNIPAM. The hydrogels were biodegradable and non-toxic against human fibroblast cells, suggesting their considerable potential for a colon-targeted drug delivery system.


Assuntos
Quitosana/química , Portadores de Fármacos/química , Hidrogéis/química , Resinas Acrílicas/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Química Click , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Humanos , Hidrogéis/farmacologia , Concentração de Íons de Hidrogênio , Mesalamina/química , Mesalamina/metabolismo , Porosidade , Temperatura
3.
Molecules ; 26(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669256

RESUMO

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked ß-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer's disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.


Assuntos
Bioensaio/métodos , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fenômenos Biofísicos , Química Click , Ligação Proteica
4.
Molecules ; 26(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670633

RESUMO

The development of new and greener approaches to organic synthesis has been a trend in recent years. Continuing the latest publications of our team, in this work, we demonstrate the efficiency of three solvents: eucalyptol (1,8-cineole), cyclopentyl methyl ether (CPME), and 2-methyltetrahydrofuran (2-MeTHF) for the synthesis of O,S,N-heterocyclic compounds.


Assuntos
Química Click/métodos , Química Verde , Compostos Heterocíclicos/síntese química , Metais/química , Solventes/química , Compostos Heterocíclicos/química
5.
Molecules ; 26(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33573040

RESUMO

In an effort to improve and achieve biologically active anticancer agents, a novel series of 1,2,3-triazole-containing hybrids were designed and efficiently synthesized via the Cu-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of substituted-arylazides with alkyne-functionalized pyrazole-[1,2,4]-triazole hybrids. The structure geometry of these new clicked 1,2,3-triazoles was explored by density functional theory (DFT) using the B3LYP/6-311++G(d,p) level; also, the potential activity of the compounds for light absorption was simulated by time-dependent DFT calculations (TD-DFT). The antitumor impacts of the newly synthesized compounds were in vitro estimated to be towards the human liver cancer cell line (HepG-2), the human colon cancer cell line (HCT-116), and human breast adenocarcinoma (MCF-7). Among the tested compounds, conjugate 7 was the most potent cytotoxic candidate towards HepG-2, HCT-116, and MCF-7, with IC50 = 12.22, 14.16, and 14.64 µM, respectively, in comparison to that exhibited by the standard drug doxorubicin (IC50 = 11.21, 12.46, and 13.45 µM). Finally, a molecular docking study was conducted within the epidermal growth factor receptor (EGFR) active site to suggest possible binding modes. Hence, it could conceivably be hypothesized that analogies 7, 6, and 5 could be considered as decent lead candidate compounds for anticancer agents.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Triazóis/química , Antineoplásicos/síntese química , Antineoplásicos/química , Química Click , Reação de Cicloadição , Teoria da Densidade Funcional , Receptores ErbB/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/farmacologia
6.
ACS Appl Mater Interfaces ; 13(3): 4711-4722, 2021 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-33444000

RESUMO

Realization of robust and facile surface functionalization processes is critical to biomaterials and biotechnology yet remains a challenge. Here, we report a new chemical approach that enables operationally simple and site-specific surface functionalization. The mechanism involves a catechol-copper redox chemistry, where the oxidative polymerization of an alkynyl catecholamine reduces Cu(II) to Cu(I), which in situ catalyzes a click reaction with azide-containing molecules of interest (MOIs). This process enables drop-coating and grafting of two- and three-dimensional solid surfaces in a single operation using as small as sub-microliter volumes. Generalizability of the method is shown for immobilizing MOIs of diverse structure and chemical or biological activity. Biological applications in anti-biofouling, cellular adhesion, scaffold seeding, and tissue regeneration are demonstrated, in which the activities or fates of cells are site-specifically manipulated. This work advances surface chemistry by integrating simplicity and precision with multipurpose surface functionalization.


Assuntos
Azidas/química , Materiais Biocompatíveis/química , Catecolaminas/química , Cobre/química , Células 3T3 , Animais , Azidas/síntese química , Materiais Biocompatíveis/síntese química , Incrustação Biológica/prevenção & controle , Catálise , Catecolaminas/síntese química , Química Click , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Oxirredução , Polimerização , Propriedades de Superfície
7.
Nat Protoc ; 16(2): 1193-1218, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33442052

RESUMO

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.


Assuntos
Replicação do DNA/fisiologia , DNA/análise , Química Click/métodos , DNA/genética , Replicação do DNA/genética , Desoxiuridina/análogos & derivados , Desoxiuridina/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Estreptavidina
8.
Bioresour Technol ; 324: 124689, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33450627

RESUMO

A method for specific immobilization of whole-cell with covalent bonds was developed through a click reaction between alkyne and azide groups. In this approach, magnetic nanoparticle Fe3O4@SiO2-NH2-alkyne was synthesized with Fe3O4 core preparation, SiO2 coating, and alkyne functionalization on the surface. The azides were successfully integrated onto the cell surface of the recombinant E. coli harboring glycerol dehydrogenase, which was employed as the model cell. The highest immobilization yield of 83% and activity recovery of 94% were obtained under the conditions of 0.67 mg mg-1 cell-support ratio, pH 6.0, temperature 45 °C, and 20 mM Cu2+ concentration. The immobilized cell showed good reusability, which remained over 50% of initial activity after 10 cycles of utilization. Its activity was 9.7-fold higher than that of the free cell at the condition of pH 8.0 and each optimal temperature. Furthermore, the immobilized cell showed significantly higher activity, operational stability, and reusability.


Assuntos
Enzimas Imobilizadas , Nanopartículas de Magnetita , Azidas , Química Click , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Polissacarídeos , Dióxido de Silício
9.
Anal Chem ; 93(4): 2610-2618, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33470097

RESUMO

Mass-spectrometry-based chemoproteomics has enabled the rapid and proteome-wide discovery of functional and potentially 'druggable' hotspots in proteins. While numerous transformations are now available, chemoproteomic studies still rely overwhelmingly on copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or 'click' chemistry. The absence of bio-orthogonal chemistries that are functionally equivalent and complementary to CuAAC for chemoproteomic applications has hindered the development of multiplexed chemoproteomic platforms capable of assaying multiple amino acid side chains in parallel. Here, we identify and optimize Suzuki-Miyaura cross-coupling conditions for activity-based protein profiling and mass-spectrometry-based chemoproteomics, including for target deconvolution and labeling site identification. Uniquely enabled by the observed orthogonality of palladium-catalyzed cross-coupling and CuAAC, we combine both reactions to achieve dual labeling. Multiplexed targeted deconvolution identified the protein targets of bifunctional cysteine- and lysine-reactive probes.


Assuntos
Alquinos/química , Azidas/química , Cobre/química , Reação de Cicloadição/métodos , Proteômica/métodos , Catálise , Química Click , Células HEK293 , Humanos , Estrutura Molecular
10.
Anal Chem ; 93(4): 2694-2705, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33397101

RESUMO

Glycoproteins secreted by cells play essential roles in the regulation of extracellular activities. Secreted glycoproteins are often reflective of cellular status, and thus glycoproteins from easily accessible bodily fluids can serve as excellent biomarkers for disease detection. Cultured cells have been extensively employed as models in the research fields of biology and biomedicine, and global analysis of glycoproteins secreted from these cells provides insights into cellular activities and glycoprotein functions. However, comprehensive identification and quantification of secreted glycoproteins is a daunting task because of their low abundances compared with the high-abundance serum proteins required for cell growth and proliferation. Several studies employed serum-free media to analyze secreted proteins, but it has been shown that serum starvation, even for a short period of time, can alter protein secretion. To overcome these issues, we developed a method to globally characterize secreted glycoproteins and their N-glycosylation sites from cultured cells by combining selective enrichment of secreted glycoproteins with a boosting approach. The results demonstrated the importance of the boosting sample selection and the boosting-to-sample ratio for improving the coverage of secreted glycoproteins. The method was applied to globally quantify secreted glycoproteins from THP-1 monocytes and macrophages in response to lipopolysaccharides (LPS) and from Hep G2 cells treated with TGF-ß without serum starvation. We found differentially secreted glycoproteins in these model systems that showed the cellular response to the immune activation or the epithelial-to-mesenchymal transition. Benefiting from the selective enrichment and the signal enhancement of low-abundance secreted glycoproteins, this method can be extensively applied to study secreted glycoproteins without serum starvation, which will provide a better understanding of protein secretion and cellular activity.


Assuntos
Glicoproteínas/química , Técnicas de Cultura de Células , Química Click , Glicoproteínas/metabolismo , Células Hep G2 , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Peptídeos/química , Fator de Crescimento Transformador beta/farmacologia
11.
Molecules ; 26(1)2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33466477

RESUMO

Continued expansion of the chemical biology toolbox presents many new and diverse opportunities to interrogate the fundamental molecular mechanisms driving complex plant-microbe interactions. This review will examine metabolic labeling with click chemistry reagents and activity-based probes for investigating the impacts of plant-associated microbes on plant growth, metabolism, and immune responses. While the majority of the studies reviewed here used chemical biology approaches to examine the effects of pathogens on plants, chemical biology will also be invaluable in future efforts to investigate mutualistic associations between beneficial microbes and their plant hosts.


Assuntos
Interações Hospedeiro-Patógeno , Microbiota , Fenômenos Fisiológicos Vegetais , Plantas/metabolismo , Plantas/microbiologia , Química Click
12.
Methods Mol Biol ; 2219: 163-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33074540

RESUMO

Many species of aquatic worms, including members of the phyla Nemertea, Annelida, Platyhelminthes, and Xenacoelomorpha, can regenerate large parts of their body after amputation. In most species, cell proliferation plays key roles in the reconstruction of lost tissues. For example, in annelids and flatworms, inhibition of cell proliferation by irradiation or chemicals prevents regeneration. Cell proliferation also plays crucial roles in growth, body patterning (e.g., segmentation) and asexual reproduction in many groups of aquatic worms. Cell proliferation dynamics in these organisms can be studied using immunohistochemical detection of proteins expressed during proliferation-associated processes or by incorporation and labeling of thymidine analogues during DNA replication. In this chapter, we present protocols for labeling and quantifying cell proliferation by (a) antibody-based detection of either phosphorylated histone H3 during mitosis or proliferating cell nuclear antigen (PCNA) during S-phase, and (b) incorporation of two thymidine analogues, 5'-bromo-2'-deoxyuridine (BrdU) and 5'-ethynyl-2'-deoxyuridine (EdU), detected by immunohistochemistry or inorganic "click" chemistry, respectively. Although these protocols have been developed for whole mounts of small (<2 cm) marine and freshwater worms, they can also be adapted for use in larger specimens or tissue sections.


Assuntos
Anelídeos/fisiologia , Platelmintos/fisiologia , Animais , Anelídeos/citologia , Ciclo Celular , Proliferação de Células , Química Click/métodos , Imuno-Histoquímica/métodos , Platelmintos/citologia , Regeneração , Fixação de Tecidos/métodos
13.
Methods Mol Biol ; 2192: 159-181, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230773

RESUMO

Human mitochondria contain their own DNA (mtDNA) that encodes 13 proteins all of which are core subunits of oxidative phosphorylation (OXPHOS) complexes. To form functional complexes, these 13 components need to be correctly assembled with approximately 70 nuclear-encoded subunits that are imported following synthesis in the cytosol. How this complicated coordinated translation and assembly is choreographed is still not clear. Methods are being developed to determine whether all members of a particular complex are translated in close proximity, whether protein synthesis is clustered in submitochondrial factories, whether these align with incoming polypeptides, and if there is evidence for co-translational translation that is regulated and limited by the interaction of the incoming proteins with synthesis of their mtDNA-encoded partners. Two methods are described in this chapter to visualize the distribution of mitochondrial ribosomal RNAs in conjunction with newly synthesized mitochondrial proteins. The first combines RNA Fluorescent In Situ Hybridization (FISH) and super-resolution immunocytochemistry to pinpoint mitochondrial ribosomal RNA. The second localizes nascent translation within the mitochondrial network through non-canonical amino acid labeling, click chemistry and fluorescent microscopy.


Assuntos
Química Click/métodos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , RNA Mitocondrial/metabolismo , RNA Ribossômico/metabolismo , Aminoácidos/química , Linhagem Celular Tumoral , DNA Mitocondrial/genética , Humanos , Microscopia de Fluorescência/métodos , Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo
14.
Methods Mol Biol ; 2254: 239-249, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33326079

RESUMO

From high-throughput DNA and RNA sequencing technologies, it is evident that more than two-thirds of the mammalian genome is transcribed and nearly 98% of the transcriptional output in humans constitute noncoding RNA, comprising tens of thousands of small and long noncoding RNAs. These observations have put the study of RNA expression levels at the center of molecular biology research. The transcriptional output of cells changes temporally throughout different cell cycle phases, or in response to a large panel of stimuli. In such instances, the measure of induced RNA transcripts might be obscured by the presence of steady-state RNA levels in the total transcriptome. With this protocol, we provide a method for labeling and purification of the nascent RNAs transcribed over short periods of time in cultured cells. The supplementation of cell culture medium with a chemically modified analog of uridine, ethynyl-uridine, allows for the subsequent biotinylation of ethynyl-uridine residues with a click-chemistry reaction. The labeled RNA is then purified on streptavidin beads and eluted. The purified RNA is suitable for use in RT-qPCR assays as well as in deep sequencing applications.


Assuntos
Técnicas de Cultura de Células/métodos , Perfilação da Expressão Gênica/métodos , RNA/química , RNA/isolamento & purificação , Ciclo Celular , Química Click , Meios de Cultura/química , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Fase S , Coloração e Rotulagem , Uridina/análogos & derivados
15.
Methods Mol Biol ; 2230: 357-365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33197025

RESUMO

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.


Assuntos
Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/ultraestrutura , Proliferação de Células/efeitos dos fármacos , Coloração e Rotulagem/métodos , Animais , Osso e Ossos/efeitos dos fármacos , Química Click/métodos , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Citometria de Fluxo/métodos , Camundongos
16.
Methods Mol Biol ; 2213: 147-161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33270200

RESUMO

Interdisciplinary chemical proteomics approaches have been widely applied to the identification of specific targets of bioactive small molecules or drugs. In this chapter, we describe the application of a cell-permeable activity-based curcumin probe (Cur-P) with an alkyne moiety to detect and identify specific binding targets of curcumin in HCT116 colon cancer cells. Through click chemistry, a fluorescent tag or a biotin tag is attached to the probe-modified curcumin targets for visualization or affinity purification followed by mass spectrometric identification. A quantitative proteomics approach of isobaric tags for relative and absolute quantification (iTRAQ)™ is applied to distinguish specific curcumin targets from nonspecific binding proteins.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Proteômica/métodos , Cromatografia por Troca Iônica , Cromatografia Líquida , Química Click , Eletroforese em Gel de Poliacrilamida , Fluorescência , Células HCT116 , Humanos , Marcação por Isótopo , Nanotecnologia , Peptídeos/metabolismo , Rodaminas , Estreptavidina/química , Espectrometria de Massas em Tandem , Tripsina/metabolismo
17.
Anal Chem ; 93(3): 1620-1626, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33369403

RESUMO

A versatile single magnetic nanoparticle (MNP)-confined, click chemistry-actuated digital DNA walker (ddWalker) machine is devised for the absolute quantification of microRNA (miRNA). This delicate ddWalker allows one target molecule to fix one DNA walking leg on a single MNP, following the Poisson statistics through a specific click chemical DNA ligation, which will initiate single molecule DNA walking to stepwise cleave the molecular beacon tracks strictly constrained on the leg-hold MNP without cross-particle reaction, fluorescently "lighting up" the exact MNP. Accordingly, the initial miRNA input can be digitally and faithfully reflected by the number of fluorescent-positive MNPs counted by a total internal reflection fluorescent microscope, enabling the absolute and precise miRNA quantification down to the femtomolar level without external calibration. This flexible ddWalker design provides a new digital signaling concept and elegantly expands the toolbox for digital biosensing.


Assuntos
DNA/química , Nanopartículas de Magnetita/química , MicroRNAs/análise , Técnicas Biossensoriais , Química Click , Células HCT116 , Humanos , Tamanho da Partícula , Propriedades de Superfície
18.
Biomolecules ; 10(12)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33322557

RESUMO

Multivalent antibody constructs have a broad range of clinical and biotechnological applications. Nanobodies are especially useful as components for multivalent constructs as they allow increased valency while maintaining a small molecule size. We here describe a novel, rapid method for the generation of bi- and multivalent nanobody constructs with oriented assembly by Cu-free strain promoted azide-alkyne click chemistry (SPAAC). We used sortase A for ligation of click chemistry functional groups site-specifically to the C-terminus of nanobodies before creating C-to-C-terminal nanobody fusions and 4-arm polyethylene glycol (PEG) tetrameric nanobody constructs. We demonstrated the viability of this approach by generating constructs with the SARS-CoV-2 neutralizing nanobody Ty1. We compared the ability of the different constructs to neutralize SARS-CoV-2 pseudotyped virus and infectious virus in neutralization assays. The generated dimers neutralized the virus similarly to a nanobody-Fc fusion variant, while a 4-arm PEG based tetrameric Ty1 construct dramatically enhanced neutralization of SARS-CoV-2, with an IC50 in the low picomolar range.


Assuntos
Anticorpos Neutralizantes/imunologia , /imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , /virologia , Química Click , Humanos , /patogenicidade , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/farmacologia
19.
Nat Commun ; 11(1): 5600, 2020 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-33154359

RESUMO

The α-type ADP-ribosylated peptides represent a class of important molecular tools in the field of protein ADP-ribosylation, however, they are difficult to access because of their inherent complicated structures and the lack of effective synthetic tools. In this paper, we present a biomimetic α-selective ribosylation reaction to synthesize a key intermediate, α-ADP-ribosyl azide, directly from native ß-nicotinamide adenine dinucleotide in a clean ionic liquid system. This reaction in tandem with click chemistry then offers a two-step modular synthesis of α-ADP-ribosylated peptides. These syntheses can be performed open air in eppendorf tubes, without the need for specialized instruments or training. Importantly, we demonstrate that the synthesized α-ADP-ribosylated peptides show high binding affinity and desirable stability for enriching protein partners, and reactivity in post-stage poly ADP-ribosylations. Owing to their simple chemistry and multidimensional bio-applications, the presented methods may provide a powerful platform to produce general molecular tools for the study of protein ADP-ribosylation.


Assuntos
Adenosina Difosfato Ribose/química , Materiais Biomiméticos/síntese química , Peptídeos/síntese química , ADP-Ribosilação , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Catálise , Química Click , Histonas/metabolismo , Líquidos Iônicos/química , NAD/química , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica
20.
Nat Commun ; 11(1): 4489, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895384

RESUMO

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Carcinoma Hepatocelular/diagnóstico , Detecção Precoce de Câncer/métodos , Vesículas Extracelulares/genética , Neoplasias Hepáticas/diagnóstico , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Química Click/instrumentação , Química Click/métodos , Química Computacional , Simulação por Computador , Diagnóstico Diferencial , Dimetilpolisiloxanos/química , Progressão da Doença , Detecção Precoce de Câncer/instrumentação , Feminino , Células Hep G2 , Humanos , Dispositivos Lab-On-A-Chip , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nanoestruturas/química , Nanofios/química , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
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