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1.
Methods ; 97: 35-43, 2016 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-26589188

RESUMEN

Recently, an increasing number of aptamers have been selected against biomarkers that are expressed at the surface of cells. This class of targets, mostly membrane proteins, is in close contact with the intra- and extra-cellular matrixes and their three-dimensional structures are inextricably linked to their inclusion in lipid bilayers. Therefore, although binding studies can be performed on the isolated form of these proteins, it remains crucial to measure the affinity of these aptamers in a more physiological environment, i.e., directly on living cells. Here, we describe a procedure for radioactive binding assays that can be adapted for measuring the affinity of aptamers against different cell lines. This method has been semi-automated using a liquid handling robot in order to reproducibly measure the apparent dissociation constant Kd and the apparent number of targets per cell. Relevant issues are discussed including the labeling of aptamers, the cells preparation, the incubation, the washings, the use of non-specific competitors, the data analysis and finally the reporting.


Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Proteínas de la Membrana/química , Animales , Adhesión Celular , Humanos , Células PC12 , Unión Proteica , Ratas , Técnica SELEX de Producción de Aptámeros , Sensibilidad y Especificidad , Análisis de la Célula Individual
2.
Int J Pharm ; 565: 59-63, 2019 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-31029658

RESUMEN

In this study, a "click and hybridization" strategy was developed for the functionalization of polydiacetylene micelles with a targeting aptamer ligand. Decoration of the nanocarriers with an anti-Annexin A2 sequence efficiently triggered enhanced internalization of the functionalized micelles in the MCF-7 cell line, with a marked increase compared to control micelles.


Asunto(s)
Anexina A2/genética , Aptámeros de Nucleótidos/administración & dosificación , Portadores de Fármacos/administración & dosificación , Micelas , Polímero Poliacetilénico/administración & dosificación , Transporte Biológico , Humanos , Células MCF-7
3.
Chem Commun (Camb) ; 54(29): 3613-3616, 2018 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-29577125

RESUMEN

Polydiacetylene micelles were functionalized with controlled amounts of biotin using bioorthogonal click chemistry. The biotinylated micelles were evaluated in the selective targeting of the MCF-7 cancerous cell line and were shown to be readily internalized. The efficiency of the cellular uptake was correlated to the density of grafted biotin.


Asunto(s)
Biotina/análogos & derivados , Micelas , Polímeros/química , Poliinos/química , Biotina/metabolismo , Biotinilación , Química Clic , Humanos , Células MCF-7 , Polímero Poliacetilénico , Polimerizacion , Polímeros/síntesis química , Polímeros/metabolismo , Poliinos/síntesis química , Poliinos/metabolismo , Receptores de Factores de Crecimiento/metabolismo
4.
Methods Mol Biol ; 1575: 253-272, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28255886

RESUMEN

Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.


Asunto(s)
Aptámeros de Nucleótidos/aislamiento & purificación , Proteínas de la Membrana/genética , Animales , Aptámeros de Nucleótidos/genética , Biomarcadores/análisis , Células CHO , Cricetulus , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de la Membrana/química , Conformación Proteica , Técnica SELEX de Producción de Aptámeros , Análisis de Secuencia de ADN
5.
Methods Mol Biol ; 1380: 135-50, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26552822

RESUMEN

Fluorescence imaging techniques could be used in different ways to study the interaction of aptamers with biological systems from cell culture to animal models. Here, we present the methods developed in our laboratory for fluorescently labeled aptamers, study their internalization inside living cells using time-lapse microscopy, and monitor their biodistribution in mice bearing subcutaneous xenograft tumors using planar fluorescence imaging and fluorescence diffuse optical tomography (fDOT).


Asunto(s)
Aptámeros de Nucleótidos , Colorantes Fluorescentes , Imagen Molecular/métodos , Animales , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Colorantes Fluorescentes/química , Xenoinjertos , Humanos , Imagenología Tridimensional/métodos , Técnicas In Vitro , Ratones , Ratones Desnudos , Microscopía por Video/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Coloración y Etiquetado , Imagen de Lapso de Tiempo/métodos , Tomografía Óptica/métodos
6.
PLoS One ; 9(1): e87002, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24489826

RESUMEN

BACKGROUND: Cell-SELEX is now widely used for the selection of aptamers against cell surface biomarkers. However, despite negative selection steps using mock cells, this method sometimes results in aptamers against undesirable targets that are expressed both on mock and targeted cells. Studying these junk aptamers might be useful for further applications than those originally envisaged. METHODOLOGY/PRINCIPAL FINDINGS: Cell-SELEX was performed to identify aptamers against CHO-K1 cells expressing human Endothelin type B receptor (ETBR). CHO-K1 cells were used for negative selection of aptamers. Several aptamers were identified but no one could discriminate between both cell lines. We decided to study one of these aptamers, named ACE4, and we identified that it binds to the Annexin A2, a protein overexpressed in many cancers. Radioactive binding assays and flow cytometry demonstrated that the aptamer was able to bind several cancer cell lines from different origins, particularly the MCF-7 cells. Fluorescence microscopy revealed it could be completely internalized in cells in 2 hours. Finally, the tumor targeting of the aptamer was evaluated in vivo in nude mice xenograft with MCF-7 cells using fluorescence diffuse optical tomography (fDOT) imaging. Three hours after intravenous injection, the aptamer demonstrated a significantly higher uptake in the tumor compared to a scramble sequence. CONCLUSIONS/SIGNIFICANCE: Although aptamers could be selected during cell-SELEX against other targets than those initially intended, they represent a potential source of ligands for basic research, diagnoses and therapy. Here, studying such aptamers, we identify one with high affinity for Annexin A2 that could be a promising tool for biomedical application.


Asunto(s)
Adenocarcinoma/diagnóstico , Anexina A2/metabolismo , Aptámeros de Nucleótidos , Neoplasias de la Mama/diagnóstico , Proteínas de Neoplasias/metabolismo , Adenocarcinoma/genética , Animales , Anexina A2/genética , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/aislamiento & purificación , Neoplasias de la Mama/genética , Células CHO , Cricetulus , Endocitosis , Femenino , Citometría de Flujo , Expresión Génica , Humanos , Hallazgos Incidentales , Ligandos , Células MCF-7 , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Unión Proteica , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Técnica SELEX de Producción de Aptámeros
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