Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 147
Filtrar
Más filtros

Banco de datos
País/Región como asunto
Tipo del documento
Intervalo de año de publicación
1.
Int J Mol Sci ; 25(9)2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38731860

RESUMEN

The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.


Asunto(s)
Aptámeros de Nucleótidos , COVID-19 , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Aptámeros de Nucleótidos/metabolismo , Aptámeros de Nucleótidos/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/efectos de los fármacos , Humanos , COVID-19/virología , COVID-19/metabolismo , Interferometría/métodos , Citometría de Flujo/métodos
2.
Nat Immunol ; 12(9): 898-907, 2011 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-21841785

RESUMEN

Regulatory T cells (T(reg) cells) are essential for self-tolerance and immune homeostasis. Lack of effector T cell (T(eff) cell) function and gain of suppressive activity by T(reg) cells are dependent on the transcriptional program induced by Foxp3. Here we report that repression of SATB1, a genome organizer that regulates chromatin structure and gene expression, was crucial for the phenotype and function of T(reg) cells. Foxp3, acting as a transcriptional repressor, directly suppressed the SATB1 locus and indirectly suppressed it through the induction of microRNAs that bound the SATB1 3' untranslated region. Release of SATB1 from the control of Foxp3 in T(reg) cells caused loss of suppressive function, establishment of transcriptional T(eff) cell programs and induction of T(eff) cell cytokines. Our data support the proposal that inhibition of SATB1-mediated modulation of global chromatin remodeling is pivotal for maintaining T(reg) cell functionality.


Asunto(s)
Ensamble y Desensamble de Cromatina/inmunología , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica , Proteínas de Unión a la Región de Fijación a la Matriz/inmunología , Autotolerancia , Linfocitos T Reguladores/inmunología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Lentivirus , Activación de Linfocitos/efectos de los fármacos , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/inmunología , MicroARNs/metabolismo , MicroARNs/farmacología , Interferencia de ARN , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Autotolerancia/efectos de los fármacos , Autotolerancia/genética , Autotolerancia/inmunología , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismo , Transducción Genética
3.
Exp Dermatol ; 32(7): 965-974, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-36995066

RESUMEN

Dermatomyositis (DM) is an idiopathic inflammatory myopathy belonging to the spectrum of autoimmune connective tissue diseases. DM patients present with antinuclear antibodies against Mi-2, also known as Chromodomain-helicase-DNA-binding protein 4 (CHD4). CHD4 is upregulated in DM skin biopsies and could potentially affect DM pathophysiology as it binds endogenous DNA with a high affinity (KD = 0.2 nM ± 0.076 nM) and forms CHD4-DNA complexes. The complexes are localized in the cytoplasm of UV-radiated and transfected HaCaTs and amplify the expression of interferon (IFN) regulated genes and the amount of functional CXCL10 protein stronger than DNA alone. The enhancement of the type I IFN pathway activation in HaCaTs through CHD4-DNA signalling suggests a possible mechanism for the sustainment of the pro-inflammatory vicious cycle in DM skin lesions.


Asunto(s)
Complejo Antígeno-Anticuerpo , Dermatomiositis , Humanos , Autoantígenos/genética , ADN , ADN Helicasas/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2
4.
Chemistry ; 29(3): e202202376, 2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36194523

RESUMEN

The glmS ribozyme regulates the expression of the essential GlmS enzyme being involved in cell wall biosynthesis. While >450 variants of the glmS ribozyme were identified by in silico approaches and homology searches, only a few have yet been experimentally investigated. Herein, we validate and characterize the glmS ribozymes of the human pathogens Clostridium difficile and Listeria monocytogenes. Both ribozymes, as their previous characterized homologs rely on glucosamine-6-phosphate as co-factor and the presence of divalent cations for exerting the cleavage reaction. The observed EC50 values in turn were found to be in the submicromolar range, at least an order of magnitude lower than observed for glmS ribozymes from other bacteria. The glmS ribozyme of L. monocytogenes was further shown to bear unique properties. It discriminates between co-factors very stringently and other than the glmS ribozyme of C. difficile retains activity at low temperatures. This finding illustrates that albeit being highly conserved, glmS ribozymes have unique characteristics.


Asunto(s)
Proteínas Bacterianas , Clostridioides difficile , Listeria monocytogenes , ARN Catalítico , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Listeria monocytogenes/genética , Listeria monocytogenes/metabolismo , ARN Catalítico/genética , ARN Catalítico/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
5.
Chemistry ; 29(3): e202202378, 2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36326082

RESUMEN

Riboswitches are 5'-untranslated mRNA regions mostly found in bacteria. They are promising drug targets to overcome emerging bacterial resistance against commonly used antibiotics. The glmS riboswitch is unique among the family of riboswitches as it is a ribozyme that undergoes self-cleavage upon binding to glucosamine-6-phosphate (GlcN6P). Previously, we showed that carba glucosamine-6-phosphate (carba-GlcN6P) induces self-cleavage of the riboswitch with a potency similar to that of GlcN6P. Here, we report a synthetic approach to a new class of carba-GlcN6P derivatives with an alkoxy substituent in the carba position. Key features of the synthesis are a ring closing metathesis followed by a hydroboration. The strategy gives access to libraries of carba-GlcN6P derivatives. Ribozyme cleavage assays unraveled new activators for the glmS riboswitch from Listeria monocytogenes and Clostridium difficile.


Asunto(s)
Carba-azúcares , ARN Catalítico , Riboswitch , ARN Catalítico/metabolismo , Carba-azúcares/metabolismo , Proteínas Bacterianas/metabolismo , Glucosamina , Fosfatos
6.
Nucleic Acids Res ; 48(8): 4013-4027, 2020 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-31989173

RESUMEN

Libraries of single-stranded oligodeoxynucleotides (ssODNs) can be enriched for sequences that specifically bind molecules on naïve complex biological samples like cells or tissues. Depending on the enrichment strategy, the ssODNs can identify molecules specifically associated with a defined biological condition, for example a pathological phenotype, and thus are potentially useful for biomarker discovery. We performed ADAPT, a variant of SELEX, on exosomes secreted by VCaP prostate cancer cells. A library of ∼1011 ssODNs was enriched for those that bind to VCaP exosomes and discriminate them from exosomes derived from LNCaP prostate cancer cells. Next-generation sequencing (NGS) identified the best discriminating ssODNs, nine of which were resynthesized and their discriminatory ability confirmed by qPCR. Affinity purification with one of the sequences (Sequence 7) combined with LC-MS/MS identified its molecular target complex, whereof most proteins are part of or associated with the multiprotein ESCRT complex participating in exosome biogenesis. Within this complex, YBX1 was identified as the directly-bound target protein. ADAPT thus is able to differentiate exosomes from cancer cell subtypes from the same lineage. The composition of ESCRT complexes in exosomes from VCaP versus LNCaP cells might constitute a discriminatory element between these prostate cancer subtypes.


Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Exosomas/metabolismo , Neoplasias de la Próstata/química , Aptámeros de Nucleótidos , Línea Celular Tumoral , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Masculino , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/metabolismo , Técnica SELEX de Producción de Aptámeros , Proteína 1 de Unión a la Caja Y/metabolismo
7.
Nat Chem Biol ; 15(11): 1085-1092, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31451761

RESUMEN

Sensory photoreceptor proteins underpin light-dependent adaptations in nature and enable the optogenetic control of organismal behavior and physiology. We identified the bacterial light-oxygen-voltage (LOV) photoreceptor PAL that sequence-specifically binds short RNA stem loops with around 20 nM affinity in blue light and weaker than 1 µM in darkness. A crystal structure rationalizes the unusual receptor architecture of PAL with C-terminal LOV photosensor and N-terminal effector units. The light-activated PAL-RNA interaction can be harnessed to regulate gene expression at the RNA level as a function of light in both bacteria and mammalian cells. The present results elucidate a new signal-transduction paradigm in LOV receptors and conjoin RNA biology with optogenetic regulation, thereby paving the way toward hitherto inaccessible optoribogenetic modalities.


Asunto(s)
Luz , Biosíntesis de Proteínas , ARN/metabolismo , Proteínas Bacterianas/metabolismo , Unión Proteica , Transducción de Señal
8.
Nucleic Acids Res ; 47(22): 11861-11879, 2019 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-31732746

RESUMEN

Nonsense-mediated decay (NMD) plays a fundamental role in the degradation of premature termination codon (PTC)-containing transcripts, but also regulates the expression of functional transcripts lacking PTCs, although such 'non-canonical' functions remain ill-defined and require the identification of factors targeting specific mRNAs to the NMD machinery. Our work identifies the stem cell-specific mRNA repressor protein TRIM71 as one of these factors. TRIM71 plays an essential role in embryonic development and is linked to carcinogenesis. For instance, TRIM71 has been correlated with advanced stages and poor prognosis in hepatocellular carcinoma. Our data shows that TRIM71 represses the mRNA of the cell cycle inhibitor and tumor suppressor CDKN1A/p21 and promotes the proliferation of HepG2 tumor cells. CDKN1A specific recognition involves the direct interaction of TRIM71 NHL domain with a structural RNA stem-loop motif within the CDKN1A 3'UTR. Importantly, CDKN1A repression occurs independently of miRNA-mediated silencing. Instead, the NMD factors SMG1, UPF1 and SMG7 assist TRIM71-mediated degradation of CDKN1A mRNA, among other targets. Our data sheds light on TRIM71-mediated target recognition and repression mechanisms and uncovers a role for this stem cell-specific factor and oncogene in non-canonical NMD, revealing the existence of a novel mRNA surveillance mechanism which we have termed the TRIM71/NMD axis.


Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Degradación de ARNm Mediada por Codón sin Sentido/fisiología , Estabilidad del ARN , Proteínas de Motivos Tripartitos/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Regiones no Traducidas 3' , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Unión Proteica , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/fisiología , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética
9.
Angew Chem Int Ed Engl ; 60(18): 10279-10285, 2021 04 26.
Artículo en Inglés | MEDLINE | ID: mdl-33683787

RESUMEN

The receptor binding domain (RBD) of the spike glycoprotein of the coronavirus SARS-CoV-2 (CoV2-S) binds to the human angiotensin-converting enzyme 2 (ACE2) representing the initial contact point for leveraging the infection cascade. We used an automated selection process and identified an aptamer that specifically interacts with CoV2-S. The aptamer does not bind to the RBD of CoV2-S and does not block the interaction of CoV2-S with ACE2. Nevertheless, infection studies revealed potent and specific inhibition of pseudoviral infection by the aptamer. The present study opens up new vistas in developing SARS-CoV2 infection inhibitors, independent of blocking the ACE2 interaction of the virus, and harnesses aptamers as potential drug candidates and tools to disentangle hitherto inaccessible infection modalities, which is of particular interest in light of the increasing number of escape mutants that are currently being reported.


Asunto(s)
Antivirales/farmacología , Aptámeros de Nucleótidos/farmacología , Tratamiento Farmacológico de COVID-19 , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/química , Aptámeros de Nucleótidos/química , Sitios de Unión/efectos de los fármacos , COVID-19/metabolismo , Descubrimiento de Drogas , Células HEK293 , Humanos , Unión Proteica/efectos de los fármacos , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , SARS-CoV-2/química , SARS-CoV-2/fisiología , Técnica SELEX de Producción de Aptámeros , Glicoproteína de la Espiga del Coronavirus/química
10.
Angew Chem Int Ed Engl ; 60(41): 22441-22446, 2021 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-34293228

RESUMEN

Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood-restoring the blood's natural coagulation capability.


Asunto(s)
Anticoagulantes/farmacología , Benzotiazoles/farmacología , Coagulación Sanguínea/efectos de los fármacos , Carbocianinas/farmacología , Luz , Anticoagulantes/química , Benzotiazoles/química , Carbocianinas/química , Humanos , Estructura Molecular
11.
Methods ; 161: 3-9, 2019 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-31152781

RESUMEN

Aptamer selection is a laborious procedure, requiring expertise and significant resources. These characteristics limit the accessibility of researchers to these molecular tools. We describe a selection procedure, making use of a robotic system that allows the fully automated selection of RNA and 2'deoxy-2'-fluoro pyrimidine RNA aptamers. The platform offers a rapid access to aptamers for basic research and development, therefore opening the path to aptamer-based systemic analysis of proteomes in biological settings.


Asunto(s)
Aptámeros de Nucleótidos/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Robótica/métodos , Aptámeros de Nucleótidos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Pirimidinas/metabolismo , ARN/metabolismo , Robótica/instrumentación
12.
Angew Chem Int Ed Engl ; 59(50): 22414-22418, 2020 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-32865316

RESUMEN

The investigation and manipulation of cellular processes with subcellular resolution requires non-invasive tools with spatiotemporal precision and reversibility. Building on the interaction of the photoreceptor PAL with an RNA aptamer, we describe a variation of the CRISPR/dCAS9 system for light-controlled activation of gene expression. This platform significantly reduces the coding space required for genetic manipulation and provides a strong on-switch with almost no residual activity in the dark. It adds to the current set of modular building blocks for synthetic biological circuit design and is broadly applicable.


Asunto(s)
Aptámeros de Nucleótidos/genética , Sistemas CRISPR-Cas/genética , Luz , Expresión Génica , Humanos , Activación Transcripcional/genética
13.
Mol Ther ; 26(1): 95-104, 2018 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-29103909

RESUMEN

The chemokine CCL17, mainly produced by dendritic cells (DCs) in the immune system, is involved in the pathogenesis of various inflammatory diseases. As a ligand of CCR4, CCL17 induces chemotaxis and facilitates T cell-DC interactions. We report the identification of two novel RNA aptamers, which were validated in vitro and in vivo for their capability to neutralize CCL17. Both aptamers efficiently inhibited the directed migration of the CCR4+ lymphoma line BW5147.3 toward CCL17 in a dose-dependent manner. To study the effect of these aptamers in vivo, we used a murine model of contact hypersensitivity. Systemic application of the aptamers significantly prevented ear swelling and T cell infiltration into the ears of sensitized mice after challenge with the contact sensitizer. The results of this proof-of-principle study establish aptamers as potent inhibitors of CCL17-mediated chemotaxis. Potentially, CCL17-specific aptamers may be used therapeutically in humans to treat or prevent allergic and inflammatory diseases.


Asunto(s)
Aptámeros de Nucleótidos/genética , Quimiocina CCL17/genética , Quimiotaxis/genética , Quimiotaxis/inmunología , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Aptámeros de Nucleótidos/química , Movimiento Celular/genética , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Conformación de Ácido Nucleico , Técnica SELEX de Producción de Aptámeros
14.
Angew Chem Int Ed Engl ; 58(31): 10752-10755, 2019 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-31050104

RESUMEN

Biomedical sciences require effective tools to manipulate, detect, and study biological phenomena. Oligo(deoxy)nucleotide ligands represent such tools, but the current strategies to generate them are restricted. Their limited availability is insufficient to address the broad range of targets related to biomedical research. Exemplified by targeting the hydrophobic molecule (-)-Δ9 -tetrahydrocannabinol (THC), we report a receptor-guided design (RGD) strategy to generate chemically modified oligodeoxynucleotide libraries for the tailored selection of clickmers.

15.
J Neurosci ; 37(10): 2517-2523, 2017 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-28275062

RESUMEN

Aptamers are short nucleic acids that interact with a variety of targets with high affinity and specificity. They have been shown to inhibit biological functions of cognate target proteins, and they are identifiable by an in vitro selection process, also termed SELEX (Systematic Evolution of Ligands by EXponential enrichment). Being nucleic acids, aptamers can be synthesized chemically or enzymatically. The latter renders RNA aptamers compatible with the cell's own transcription machinery and, thus, expressable inside cells. The synthesis of aptamers by chemical approaches opens up the possibility of producing aptamers on a large scale and enables a straightforward access to introduce modifications in a site-specific manner (e.g., fluorophores or photo-labile groups). These characteristics make aptamers broadly applicable (e.g., as an analytical, diagnostic, or separation tool). In this TechSight, we provide a brief overview on aptamer technology and the potential of aptamers as valuable research tools in neurosciences.


Asunto(s)
Aptámeros de Nucleótidos/genética , Marcación de Gen/tendencias , Neuronas/fisiología , Neurociencias/tendencias , Patología Molecular/tendencias , Técnica SELEX de Producción de Aptámeros/tendencias , Animales , Ingeniería Genética/tendencias , Humanos
16.
J Proteome Res ; 17(6): 2174-2181, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29703078

RESUMEN

Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers' ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for "fishing out" aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer-target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by reverse-phase chromatography combined with fluorescence detection followed by mass-spectrometry-based protein identification. These comparative results of several methodological approaches confirmed the universal applicability of this method to studying aptamer-protein interactions with high sensitivity, showing superior properties compared with pull-down techniques.


Asunto(s)
Aptámeros de Péptidos/metabolismo , Fluoresceínas , Técnica SELEX de Producción de Aptámeros/métodos , Cromatografía , Electroforesis , Colorantes Fluorescentes , Humanos , Espectrometría de Masas , Métodos , Unión Proteica
17.
Chembiochem ; 18(5): 435-440, 2017 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-28012261

RESUMEN

The ever-growing number of pathogenic bacteria resistant to treatment with antibiotics call for the development of novel compounds with as-yet unexplored modes of action. Here, we demonstrate the in vivo antibacterial activity of carba-α-d-glucosamine (CGlcN). In this mode of action study, we provide evidence that CGlcN-mediated growth inhibition is due to glmS ribozyme activation, and we demonstrate that CGlcN hijacks an endogenous activation pathway, hence utilizing a prodrug mechanism. This is the first report describing antibacterial activity mediated by activating the self-cleaving properties of a ribozyme. Our results open the path towards a compound class with an entirely novel and distinct molecular mechanism.


Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Ciclohexanoles/química , Ciclohexilaminas/química , Glucosamina/farmacología , ARN Catalítico/metabolismo , Antibacterianos/química , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Activación Enzimática/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Mutación , ARN Catalítico/genética
18.
Chemistry ; 23(51): 12604-12612, 2017 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-28661578

RESUMEN

The glmS ribozyme is a bacterial gene-regulating riboswitch that controls cell wall synthesis, depending on glucosamine-6-phosphate as a cofactor. Due to the presence of this ribozyme in several human pathogen bacteria (e.g., MRSA, VRSA), the glmS ribozyme represents an attractive target for the development of artificial cofactors. The substitution of the ring oxygen in carbohydrates by functionalized methylene groups leads to a new generation of glycomimetics that exploits distinct interaction possibilities with their target structure in biological systems. Herein, we describe the synthesis of mono-fluoro-modified carba variants of α-d-glucosamine and ß-l-idosamine. (5aR)-Fluoro-carba-α-d-glucosamine-6-phosphate is a synthetic mimic of the natural ligand of the glmS ribozyme and is capable of effectively addressing its unique self-cleavage mechanism. However, in contrast to what was expected, the activity is significantly decreased compared to its non-fluorinated analog. By combining self-cleavage assays with the Bacillus subtilis and Staphylococcus aureus glmS ribozyme and molecular docking studies, we provide a structure-activity relationship for fluorinated carba-sugars.


Asunto(s)
Proteínas Bacterianas/metabolismo , Carba-azúcares/metabolismo , ARN Catalítico/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Carba-azúcares/síntesis química , Carba-azúcares/química , Ciclohexanoles/síntesis química , Ciclohexanoles/química , Ciclohexanoles/metabolismo , Ciclohexilaminas/síntesis química , Ciclohexilaminas/química , Ciclohexilaminas/metabolismo , Halogenación , Conformación Molecular , Simulación del Acoplamiento Molecular , Conformación de Ácido Nucleico , ARN Catalítico/química , Staphylococcus aureus/metabolismo , Estereoisomerismo , Relación Estructura-Actividad
19.
Methods ; 97: 104-9, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26615953

RESUMEN

Apta-PCR is an ultrasensitive assay in which aptamers are exploited not only as biomolecular recognition elements, but also as reporter labels for amplification via real-time PCR. This methodology has been successfully applied to the detection of proteins, achieving limits of detection in the picomolar range. The introduction of caged aptamers that bear photo-labile groups, so called cages, at strategic positions so that their tertiary structure and thus their binding properties can be controlled by light, facilitates a more robust and attractive assay in terms of sample conservation and reusability. In this work, we report for the first time the use of caged aptamers for cell detection in an apta-PCR assay. Specifically, a sandwich format is used combining the capture of B-cells by an antibody with the specific detection of Burkitt's lymphoma cancer cells by a caged aptamer, acting as a reporter probe. Elution of the aptamer bound to the cancer cells is performed by light and the number of cells is then correlated with the amount of eluted caged aptamer using real-time PCR analysis. The reported technique shows an excellent sensitivity, achieving detection of as few as 77 cells, and due to the inherent robustness of the assay, this detection platform can be reused for further analyses, demonstrating potential applicability in proteomics and clinical diagnostics.


Asunto(s)
Aptámeros de Nucleótidos/química , Neoplasias/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Línea Celular Tumoral , G-Cuádruplex , Humanos , Secuencias Invertidas Repetidas
20.
Angew Chem Int Ed Engl ; 56(1): 359-363, 2017 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-27897376

RESUMEN

Intramolecular circularization of DNA oligonucleotides was accomplished by incorporation of alkyne-modified photolabile nucleosides into DNA sequences, followed by a CuI -catalyzed alkyne-azide cycloaddition with bis-azido linker molecules. We determined a range of ring sizes, in which the caged circular oligonucleotides exhibit superior duplex destabilizing properties. Specific binding of a full-length 90 nt C10 aptamer recognizing human Burkitt's lymphoma cells was then temporarily inhibited by locking the aptamer in a bicircularized structure. Irradiation restored the native aptamer conformation resulting in efficient cell binding and uptake. The photo-tether strategy presented here provides a robust and versatile tool for the light-activation of longer functional oligonucleotides, noteworthy without prior knowledge on the structure and the importance of specific nucleotides within a DNA aptamer.


Asunto(s)
Oligonucleótidos/química , Conformación de Carbohidratos , Procesos Fotoquímicos
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA