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1.
Food Chem ; 317: 126433, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092613

RESUMO

Highly catalytic and stable N-doped carbon dots (N-CDs) were prepared rapidly by microwave procedure using glucose as precursor and ammonium sulfite as N-dopant. The reduction of AgNO3 by trisodium citrate (TCA) was slow to form nanosilver (AgNP), and the N-CDs exhibited strong catalysis of the AgNP reaction. The formed AgNPs were used as indicator in the presence of Vitoria blue B (VBB) molecule probe with a SERS peak at 1615 cm-1. With the increase of nancatalyst N-CDs concentration, the AgNP reaction speed up, and the SERS peak of VBB enhanced linearly due to formation of more AgNPs as substrate. In the presence of avidin (Ad), the SERS peak weakened. Upon addition of biotin, the SERS peak enhanced due to turn on the indicator nanoreaction. The enhanced SERS signal had a good linear relationship with the biotin concentration in range of 0.0006-0.021 ng/mL, with a detection limit of 0.3 pg/mL.


Assuntos
Biotina/análise , Análise de Alimentos/métodos , Prata/química , Análise Espectral Raman/métodos , Animais , Avidina/química , Carbono/química , Catálise , Citratos/química , Análise de Alimentos/instrumentação , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Sonda Molecular/instrumentação , Sondas Moleculares/química , Compostos Orgânicos/química , Pontos Quânticos/química , Nitrato de Prata/química , Análise Espectral Raman/instrumentação
2.
Yakugaku Zasshi ; 139(12): 1531-1538, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31787640

RESUMO

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are powerful molecular imaging methods for examining disease-related factors in the whole body using specific imaging probes. Recently, we tried to develop molecular imaging probes that specifically visualize pathological factors associated with cancers and infectious diseases. Although survivin is highly expressed in several cancers, its expression is undetectable in non-dividing tissues. Thus, we developed several small molecular imaging probes that target survivin. These ligands not only showed high affinity for survivin protein, but also showed consistent cellular accumulation with respect to survivin expression levels, thereby indicating the feasibility of their backbones as scaffolds for tumor-specific imaging agents that target survivin. Prion diseases are fatal neurodegenerative diseases characterized by the deposition of amyloid plaques containing abnormal prion protein aggregates (PrPSc). Thus, we developed flavonoids, acridines, and benzofurans as PrPSc-imaging probes. A styrylchromone derivative ([123I]SC-OMe) appears to be a particularly promising SPECT radioligand for monitoring prion deposit levels in living brains. Gallium-68 is a positron emitter in clinical PET applications that can be produced by a 68Ge/68Ga generator without a cyclotron. Notably, we developed new adsorbents for 68Ge by introducing N-methylglucamine groups into the Sephadex series to serve as a hydrophilic polymer matrix. We also demonstrated that generator-eluted 68Ga-citrate could be used for PET imaging of infectious mouse models. Our polysaccharide-based 68Ge/68Ga generators were shown to be prospectively cost-effective production systems for 68Ga radiopharmaceuticals. This Review describes the major findings of these three studies and the future prospect of these fields.


Assuntos
Doenças Transmissíveis/diagnóstico por imagem , Imagem Molecular/métodos , Técnicas de Sonda Molecular , Sondas Moleculares , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Acridinas , Animais , Benzofuranos , Flavonoides , Radioisótopos de Gálio , Humanos , Camundongos , Neoplasias/metabolismo , Compostos Radiofarmacêuticos , Survivina/metabolismo
3.
Nat Commun ; 10(1): 4526, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586057

RESUMO

Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.


Assuntos
Citosol/química , Peróxido de Hidrogênio/análise , Técnicas de Sonda Molecular , Peroxirredoxinas/química , Membrana Celular/química , Genes Reporter , Peróxido de Hidrogênio/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mitocôndrias/química , Sondas Moleculares/química , Oxirredução , Engenharia de Proteínas , Schizosaccharomyces
4.
J Dermatol ; 46(10): 917-921, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31392741

RESUMO

While the etiology of sarcoidosis remains uncertain, mycobacteria have been suggested as a causative infectious agent. To investigate the causal relationship between mycobacteria and sarcoidosis, we performed a reverse blot hybridization assay (REBA) to identify mycobacteria from the skin samples of nine patients with sarcoidosis. Six of the nine samples were shown to be positive for mycobacteria by REBA, including Mycobacterium tuberculosis and non-tuberculous mycobacteria. This is the first study to identify mycobacteria from the skin samples of sarcoidosis patients using REBA, and our results could strengthen the etiologic association between mycobacteria and sarcoidosis.


Assuntos
Mycobacterium tuberculosis/isolamento & purificação , Micobactérias não Tuberculosas/isolamento & purificação , Sarcoidose/microbiologia , Dermatopatias/microbiologia , Pele/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Sondas de DNA , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Sonda Molecular , Mycobacterium tuberculosis/genética , Micobactérias não Tuberculosas/genética , Reação em Cadeia da Polimerase , Sarcoidose/patologia , Pele/patologia , Dermatopatias/patologia
5.
BMC Infect Dis ; 19(1): 738, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438880

RESUMO

BACKGROUND: The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results. METHODS: 100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank. RESULTS: All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%. CONCLUSIONS: In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.


Assuntos
Técnicas de Genotipagem/métodos , Hepacivirus/genética , Hepatite C/diagnóstico , Técnicas de Sonda Molecular , Kit de Reagentes para Diagnóstico , Análise de Sequência de RNA/métodos , Regiões 5' não Traduzidas , Sequência de Bases , Comércio , Genômica/métodos , Genótipo , Técnicas de Genotipagem/economia , Hepacivirus/isolamento & purificação , Hepatite C/virologia , Humanos , Técnicas de Sonda Molecular/economia , Filogenia , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico/economia , Estudos Retrospectivos , Análise de Sequência de RNA/economia , Centros de Atenção Terciária
6.
Forensic Sci Int Genet ; 42: 227-234, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31377480

RESUMO

Next generation sequencing (NGS) technologies have enabled the possibility of analyzing a large number of SNPs simultaneously from multiple samples in a single experiment, for complementing the shortcomings of STR based methods. To efficiently genotype the desired SNPs, it is critical to optimize the library construction procedures. In this study, we formulated a strategy combining the molecular inversion probe (MIP) based target region capture method and NGS for genotyping 1245 SNPs. All the SNPs we selected exhibited high heterozygosity (minor allele frequency (MAF) > 0.3) according to 1000 genomes data. We applied the method to genotype a population of 210 unrelated individuals from the Hubei province of China and assessed the allele frequencies, Hardy-Weinberg equilibrium and linkage disequilibrium. The MAFs of more than 95% of the SNPs were ≥0.2, and no significant deviation or strong linkage was observed for 98% of the SNPs. The data indicated that, even within a relatively confined region, our SNP panel is suitable for individual identifications. Furthermore, we performed paternity test for 7 trio families using low quality DNA samples. The conclusions are in total agreement with these of STR-based analyses, with higher confidence indexes. Finally, we evaluated the performance of the MIP-NGS method with mock degraded DNA samples. We were able to genotype most of the SNPs even when the genomic DNA was sonicated to ˜100 bp range. In summary, we established a highly accurate and cost-effective method of SNP genotyping, which is potentially capable of solving complex issues encountered in forensic practices.


Assuntos
Genética Populacional , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Sonda Molecular , Polimorfismo de Nucleotídeo Único , China , Impressões Digitais de DNA , Grupos Étnicos/genética , Genética Forense/métodos , Frequência do Gene , Genótipo , Humanos
7.
Am J Physiol Endocrinol Metab ; 317(1): E74-E84, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939051

RESUMO

Intrinsically labeled dietary proteins have been used to trace various aspects of digestion and absorption, including quantifying the contribution of dietary protein to observed postprandial amino acid and protein kinetics in human subjects. Quantification of the rate of appearance in peripheral blood of an unlabeled (tracee) amino acid originating from an intrinsically labeled protein (exogenous Ra) requires the assumption that there is no dilution of the isotope enrichment of the protein-bound amino acid in the gastrointestinal tract or across the splanchnic bed. It must also be assumed that the effective volume of distribution into which the tracer and tracee appear can be reasonably estimated by a single value and that any recycling of the tracer is minimal and thus does not affect calculated rates. We have assessed these assumptions quantitatively using values from published studies. We conclude that the use of intrinsically labeled proteins as currently described to quantify exogenous Ra systematically underestimates the true value. When used with the tracer-determined rates of amino acid kinetics, underestimation of exogenous Ra from the intrinsically labeled protein method likely translates to incorrect conclusions regarding protein breakdown, including the effect of a protein meal and the anabolic impact of the speed of digestion and absorption of amino acids. Estimation of exogenous Ra from the bioavailability of ingested protein has some advantages as compared with the intrinsically labeled protein method. We therefore conclude that the bioavailability method for estimating exogenous Ra is preferable to the intrinsically labeled protein method.


Assuntos
Proteínas na Dieta/farmacocinética , Marcação por Isótopo/métodos , Proteínas/metabolismo , Imagem Corporal Total/métodos , Aminoácidos/metabolismo , Aminoácidos/farmacocinética , Disponibilidade Biológica , Deutério , Proteínas na Dieta/metabolismo , Estudos de Avaliação como Assunto , Humanos , Íleo/metabolismo , Absorção Intestinal/fisiologia , Cinética , Técnicas de Sonda Molecular , Período Pós-Prandial
8.
Glycoconj J ; 36(2): 175-183, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30993518

RESUMO

Lectins, in combination with our established enzyme-linked lectin sorbent assay (ELLSA) and inhibition study, have been used as powerful tools in many glycoconjugate recognition studies. In this short review, we highlight the following: (i) The recognition profiles of Gal/GalNAc-specific lectins were updated and upgraded. (ii) Based on the cross-specificities of applied lectins, a new classification system was introduced. (iii) Applications of lectins for the detection and identification of N-glycan and/or Tn glycotope in glycoconjugates were intergraded. (iv) The polyvalency of the glycotopes in glycans was found to play a critical role in glycan-lectin recognition. This is an unexplored area of glycobiology and one of the most promising directions toward the coming glycoscience transformation.


Assuntos
Glicômica/métodos , Lectinas/metabolismo , Animais , Humanos , Lectinas/química , Técnicas de Sonda Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Ligação Proteica
9.
Nat Commun ; 10(1): 1111, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30846702

RESUMO

Activated macrophages have the potential to be ideal targets for imaging inflammation. However, probe selectivity over non-activated macrophages and probe delivery to target tissue have been challenging. Here, we report a small molecule probe specific for activated macrophages, called CDg16, and demonstrate its application to visualizing inflammatory atherosclerotic plaques in vivo. Through a systematic transporter screen using a CRISPR activation library, we identify the orphan transporter Slc18b1/SLC18B1 as the gating target of CDg16.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Inflamação/diagnóstico por imagem , Inflamação/imunologia , Ativação de Macrófagos , Acridinas , Animais , Sistemas CRISPR-Cas , Células HeLa , Humanos , Inflamação/metabolismo , Camundongos , Camundongos Knockout para ApoE , Técnicas de Sonda Molecular , Sondas Moleculares , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Células RAW 264.7
10.
Artigo em Inglês | MEDLINE | ID: mdl-30925315

RESUMO

An efficient and novel 2,5-bis(benzo[d]thiazol-2-yl)phenol scaffold-based ratiometric fluorescent probe BTP-Cys for the sensing of cysteine has been developed. The probe BTP-Cys with acrylates moiety, as recognition site, has been successfully constructed on account of the excited state intramolecular proton transfer (ESIPT) mechanism. Upon the treatment with Cys (0-250 µM), this probe BTP-Cys exhibits a dramatic fluorescent intensity ratios enhancement (from 0.03 to 18.3) and a large emission shift (113 nm). The detection limit of this probe is as low as 3.8 × 10-7 M. Importantly, the concentration and time dependent of Cys in bovine serum albumin (BSA) has also been measured, indicating that BTP-Cys could be a biocompatible and rapid probe for Cys in vitro.


Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Fenóis/química , Soroalbumina Bovina/análise , Animais , Bovinos , Cisteína/química , Concentração de Íons de Hidrogênio , Limite de Detecção , Técnicas de Sonda Molecular , Soroalbumina Bovina/química , Espectrometria de Fluorescência
11.
Biotechnol J ; 14(7): e1800645, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30791223

RESUMO

Herein, the ribonuclease H (RNase H) activity assay based on the target-activated DNA polymerase activity is described. In this method, a detection probe composed of two functional sequences, a binding site for DNA polymerase and a catalytic substrate for RNase H, serves as a key component. The detection probe, at its initial state, suppresses the DNA polymerase activity, but it becomes destabilized by RNase H, which specifically hydrolyzes RNA in RNA/DNA hybrid duplexes. As a result, DNA polymerase recovers its activity and initiates multiple primer extension reactions in a separate TaqMan probe-based signal transduction module, leading to a significantly enhanced fluorescence "turn-on" signal. This assay can detect RNase H activity as low as 0.016 U mL-1 under optimized conditions. Furthermore, its potential use for evaluating RNase H inhibitors, which have been considered potential therapeutic agents against acquired immune deficiency syndrome (AIDS), is successfully explored. In summary, this approach is quite promising for the sensitive and accurate determination of enzyme activity and inhibitor screening.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Descoberta de Drogas/métodos , Ensaios Enzimáticos/métodos , Técnicas de Sonda Molecular , Ribonuclease H , Estabilidade Enzimática , Ribonuclease H/análise , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo
12.
Biotechnol J ; 14(5): e1800647, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30810268

RESUMO

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 n m and a dissociation rate that is 55- to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.


Assuntos
Afinidade de Anticorpos/imunologia , Imunoglobulina G/química , Imunoglobulina G/isolamento & purificação , Peptídeos/química , Domínios Proteicos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Sítios de Ligação , Sítios de Ligação de Anticorpos , Biotina , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Cisteína/química , Ensaio de Imunoadsorção Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/imunologia , Humanos , Imobilização , Proteínas Imobilizadas/química , Proteínas Imobilizadas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Cinética , Modelos Moleculares , Técnicas de Sonda Molecular , Peptídeo Hidrolases , Ligação Proteica , Especificidade por Substrato , Leveduras
13.
Curr Top Microbiol Immunol ; 420: 253-281, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30244324

RESUMO

The activity of proteases is tightly regulated, and dysregulation is linked to a variety of human diseases. For this reason, ABPP is a well-suited method to study protease biology and the design of protease probes has pushed the boundaries of ABPP. The development of highly selective protease probes is still a challenging task. After an introduction, the first section of this chapter discusses several strategies to enable detection of a single active protease species. These range from the usage of non-natural amino acids, combination of probes with antibodies, and engineering of the target proteases. A next section describes the different types of detection tags that facilitate the read-out possibilities including various types of imaging methods and mass spectrometry-based target identification. The power of protease ABPP is illustrated by examples for a selected number of proteases. It is expected that some protease probes that have been evaluated in animal models of human disease will find translation into clinical application in the near future.


Assuntos
Ensaios Enzimáticos/métodos , Técnicas de Sonda Molecular , Peptídeo Hidrolases/análise , Peptídeo Hidrolases/metabolismo , Animais , Endopeptidases/análise , Endopeptidases/química , Endopeptidases/metabolismo , Humanos , Peptídeo Hidrolases/química
14.
RNA ; 25(1): 135-146, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30389828

RESUMO

Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3' extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.


Assuntos
Carbodi-Imidas , Sondas Moleculares , RNA/química , Animais , Pareamento de Bases , Sequência de Bases , Células Cultivadas , Indicadores e Reagentes , Camundongos , Técnicas de Sonda Molecular , Estrutura Molecular , Conformação de Ácido Nucleico , RNA/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Ésteres do Ácido Sulfúrico
15.
Artigo em Inglês | MEDLINE | ID: mdl-30384021

RESUMO

A new near-infrared ratiometric type fluorescent probe was prepared. 3-formyl BODIPY derivatives without substituent group in the 2, 6-position was obtained through DDQ oxidation reaction. Furthermore, it reacted with indole salt to produce probe. This probe bears indolium group as the recognition site and the 3-formyl-BODIPY as fluorophore. The specific detection of cyanide was conducted the nucleophilic attack of cyanide toward the indolium group of the probe, breaking the intramolecular charge transfer (ICT) effect and generating a ratio change in the fluorescence signal. The probe has high selectivity and sensitivity for cyanide. Moreover, cell experiments indicated this probe was benign to HepG-2 cells, and has the potential application in imaging CN- in living HepG-2 cells.


Assuntos
Técnicas Biossensoriais/métodos , Cianetos/análise , Fluorescência , Corantes Fluorescentes/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Células Hep G2 , Humanos , Técnicas de Sonda Molecular
16.
Methods Mol Biol ; 1870: 263-271, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30539562

RESUMO

Posttranscriptional modification of mRNAs plays an important role in establishing the functional diversity of the proteome. The m6A modification is found in many species of RNA, including tRNA, mRNA, rRNA, and long noncoding RNAs. The physiological role of m6A modification of RNA is not fully explored and is a topic of current research. It is predicted that the major effect of m6A modification of mRNAs is on its stability and/or translation. The global changes in m6A levels in total RNA or particular species of RNAs can be measured by dot blot analysis using m6A specific antibodies or using mass spectrometry following chromatographic separation. The dot blot method for detection of global m6A changes is a relatively straightforward method to quantitate m6A modification but suffers from low sensitivity when the fraction of m6A-modified RNA is small in analyzed samples. Here, we describe a modified dot blot method that is sensitive and quantitative for detecting m6A-modified RNA by adding an immunoprecipitation step to enrich for m6A-modified RNA.


Assuntos
Adenosina/análogos & derivados , Técnicas de Sonda Molecular , Processamento Pós-Transcricional do RNA , RNA/genética , Interpretação Estatística de Dados , Imunoprecipitação , Metilação
17.
RNA ; 25(1): 147-157, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30341176

RESUMO

Many biological functions performed by RNAs arise from their in vivo structures. The structure of the same RNA can differ in vitro and in vivo owing in part to the influence of molecules ranging from protons to secondary metabolites to proteins. Chemical reagents that modify the Watson-Crick (WC) face of unprotected RNA bases report on the absence of base-pairing and so are of value to determining structures adopted by RNAs. Reagents have thus been sought that can report on the native RNA structures that prevail in living cells. Dimethyl sulfate (DMS) and glyoxal penetrate cell membranes and inform on RNA secondary structure in vivo through modification of adenine (A), cytosine (C), and guanine (G) bases. Uracil (U) bases, however, have thus far eluded characterization in vivo. Herein, we show that the water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is capable of modifying the WC face of U and G in vivo, favoring the former nucleobase by a factor of ∼1.5, and doing so in the eukaryote rice, as well as in the Gram-negative bacterium Escherichia coli While both EDC and glyoxal target Gs, EDC reacts with Gs in their typical neutral state, while glyoxal requires Gs to populate the rare anionic state. EDC may thus be more generally useful; however, comparison of the reactivity of EDC and glyoxal may allow the identification of Gs with perturbed pKas in vivo and genome-wide. Overall, use of EDC with DMS allows in vivo probing of the base-pairing status of all four RNA bases.


Assuntos
Etildimetilaminopropil Carbodi-Imida , RNA/química , Pareamento de Bases , Sequência de Bases , Escherichia coli/química , Escherichia coli/genética , Glioxal , Guanina/química , Indicadores e Reagentes , Técnicas de Sonda Molecular , Sondas Moleculares , Estrutura Molecular , Conformação de Ácido Nucleico , Oryza/química , Oryza/genética , RNA/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA de Plantas/química , RNA de Plantas/genética , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , RNA Ribossômico 5,8S/química , RNA Ribossômico 5,8S/genética , Uracila/química
18.
RNA ; 25(1): 82-89, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30309880

RESUMO

Many approaches exist to detect RNA using complementary oligonucleotides. DNA ligation-based techniques can improve discrimination of subtle sequence variations, but they have been difficult to implement for direct RNA analysis due to the infidelity and inefficiency of most DNA ligases on RNA. In this report, we have systematically studied if ribonucleotide substitutions in padlock probes can provide higher catalytic efficiencies for Chlorella virus DNA ligase (PBCV-1 DNA ligase) and T4 RNA ligase 2 (T4Rnl2) on RNA. We provide broad characterization of end-joining fidelity for both enzymes in RNA-templated 3'-OH RNA/5'-pDNA chimeric probe ligation. Both ligases showed increased ligation efficiency toward chimeric substrates on RNA. However, end-joining fidelity of PBCV-1 DNA ligase remained poor, while T4Rnl2 showed a somewhat better end-joining fidelity compared to PBCV-1 DNA ligase. The recently presented invader padlock (iLock) probes overcome the poor end-joining fidelity of PBCV-1 DNA ligase by the requirement of target-dependent 5' flap removal prior to ligation. Here we show that two particular ribonucleotide substitutions greatly improve the activation and ligation rate of chimeric iLock probes on RNA. We characterized the end-joining efficiency and fidelity of PBCV-1 DNA ligase and T4Rnl2 with chimeric iLock probes on RNA and found that both enzymes exhibit high ligation fidelities for single nucleotide polymorphisms on RNA. Finally, we applied the chimeric probe concept to directly differentiate between human and mouse ACTB mRNA in situ, demonstrating chimeric padlock and iLock probes as superior to their DNA equivalents.


Assuntos
Técnicas de Sonda Molecular , Sondas de Oligonucleotídeos/genética , RNA/análise , RNA/genética , Actinas/genética , Animais , Sequência de Bases , DNA Ligases/metabolismo , Humanos , Camundongos , RNA Ligase (ATP)/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Especificidade da Espécie , Especificidade por Substrato , Proteínas Virais/metabolismo
19.
Curr Top Microbiol Immunol ; 420: 233-251, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30203394

RESUMO

Protein arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine to form peptidyl citrulline. This modification is increased in multiple inflammatory diseases and in certain cancers. PADs regulate a variety of signaling pathways including apoptosis, terminal differentiation, and transcriptional regulation. Activity-based protein profiling (ABPP) probes have been developed to understand the role of the PADs in vivo and to investigate the effect of protein citrullination in various pathological conditions. Furthermore, these ABPPs have been utilized as a platform for high-throughput inhibitor discovery. This review will showcase the development of ABPPs targeting the PADs. In addition, it provides a brief overview of PAD structure and function along with recent advances in PAD inhibitor development.


Assuntos
Citrulinação , Citrulina/metabolismo , Desiminases de Arginina em Proteínas/análise , Desiminases de Arginina em Proteínas/metabolismo , Proteômica/métodos , Citrulinação/efeitos dos fármacos , Citrulina/química , Doença , Humanos , Técnicas de Sonda Molecular , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/química
20.
Nat Protoc ; 14(1): 51-67, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30487655

RESUMO

A better understanding of DNA replication initiation in human cells and how this process is altered upon DNA replication stress requires the ability to study origin firing genome wide. Previously described methods of mapping DNA replication origins in higher eukaryotes rely principally on fractionation of DNA fragments based on their size and, optionally, on the presence of ribonucleotides at their 5' end. Here, we describe a protocol for EdUseq-HU, a method for mapping early S-phase replication origins. Cells, synchronized by mitotic shake-off, are released in medium containing 5-ethynyl-2'-deoxyuridine (EdU; to label nascent DNA) and hydroxyurea (HU; to limit fork progression after origin firing). After using click chemistry to tag the EdU label with a biotin conjugate that is cleavable under mild conditions, the nascent DNA is captured on streptavidin beads. One variant of EdUseq-HU allows mapping of DNA replication origins on the genome at a resolution of 10 kb, and a second variant monitors progression of replication forks. Using EdUseq-HU, the spatiotemporal program of DNA replication in human cell lines can be interrogated in <2 weeks. The protocol requires basic cell culture and molecular biology skills, as well as familiarity with the Perl programming language and the Linux operating system.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Química Click/métodos , Replicação do DNA , DNA/genética , Técnicas de Sonda Molecular , Biotina/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Genoma Humano , Células HeLa , Humanos , Hidroxiureia/farmacologia , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Origem de Replicação , Software , Estreptavidina/química
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