RESUMEN
The advent of molecular tension probes for real-time mapping of piconewton forces in living systems has had a major impact on mechanobiology. For example, DNA-based tension probes have revealed roles for mechanics in platelet, B cell, T cell, and fibroblast function. Nonetheless, imaging short-lived forces transmitted by low-abundance receptors remains a challenge. This is a particular problem for mechanoimmunology where ligand-receptor bindings are short lived, and a few antigens are sufficient for cell triggering. Herein, we present a mechanoselection strategy that uses locking oligonucleotides to preferentially and irreversibly bind DNA probes that are mechanically strained over probes at rest. Thus, infrequent and short-lived mechanical events are tagged. This strategy allows for integration and storage of mechanical information into a map of molecular tension history. Upon addition of unlocking oligonucleotides that drive toehold-mediated strand displacement, the probes reset to the real-time state, thereby erasing stored mechanical information. As a proof of concept, we applied this strategy to study OT-1 T cells, revealing that the T cell receptor (TCR) mechanically samples antigens carrying single amino acid mutations. Such events are not detectable using conventional tension probes. Each mutant peptide ligand displayed a different level of mechanical sampling and spatial scanning by the TCR that strongly correlated with its functional potency. Finally, we show evidence that T cells transmit pN forces through the programmed cell death receptor-1 (PD1), a major target in cancer immunotherapy. We anticipate that mechanical information storage will be broadly useful in studying the mechanobiology of the immune system.
Asunto(s)
Antígenos , Sondas de ADN , Mecanotransducción Celular , Péptidos , Receptores de Antígenos de Linfocitos T , Linfocitos T , Antígenos/química , Antígenos/genética , Antígenos/inmunología , Línea Celular , Sondas de ADN/química , Sondas de ADN/genética , Sondas de ADN/inmunología , Humanos , Mecanotransducción Celular/genética , Mecanotransducción Celular/inmunología , Mutación , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/química , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
The Fragile X mental retardation-1 (Fmr1) gene encodes a multifunctional protein, FMRP, with intrinsic RNA binding activity. We have developed an approach, antibody-positioned RNA amplification (APRA), to identify the RNA cargoes associated with the in vivo configured FMRP messenger ribonucleoprotein (mRNP) complex. Using APRA as a primary screen, putative FMRP RNA cargoes were assayed for their ability to bind directly to FMRP using traditional methods of assessing RNA-protein interactions, including UV-crosslinking and filter binding assays. Approximately 60% of the APRA-defined mRNAs directly associate with FMRP. By examining a subset of these mRNAs and their encoded proteins in brain tissue from Fmr1 knockout mice, we have observed that some of these cargoes as well as the proteins they encode show discrete changes in abundance and/or differential subcellular distribution. These data are consistent with spatially selective regulation of multiple biological pathways by FMRP.
Asunto(s)
Síndrome del Cromosoma X Frágil/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN , Animales , Anticuerpos Monoclonales , Sondas de ADN/inmunología , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil/genética , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/inmunología , Purinas/metabolismo , Fracciones SubcelularesRESUMEN
The emerging nanomaterial, quantum dots or QDs, offers numerous potential applications in the biological area. As cell labeling probes, QDs become now an alternative of existing organic fluorescent dyes and fluorescent proteins. In this short review, we cover typical and successful applications of QDs as fluorescent probes in cell labeling and genomic diagnosis. As a future important application, biomolecular detection at a single molecule level utilizing QDs is also discussed.
Asunto(s)
Diagnóstico por Imagen , Nanotecnología , Proteínas/química , Animales , Fenómenos Fisiológicos Celulares , Sondas de ADN/inmunología , Colorantes Fluorescentes , Humanos , ToxicologíaRESUMEN
The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas de Homeodominio/inmunología , Regiones Promotoras Genéticas/genética , Esterol O-Aciltransferasa/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Sitios de Unión/genética , Factor de Transcripción CDX2 , Células CHO , Células CACO-2 , Cricetinae , Sondas de ADN/genética , Sondas de ADN/inmunología , Sondas de ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Glutatión Transferasa/genética , Glutatión Transferasa/inmunología , Glutatión Transferasa/metabolismo , Células HeLa , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transactivadores , Células Tumorales Cultivadas , Esterol O-Aciltransferasa 2RESUMEN
Here we demonstrate the use of redox labeled double- and single-stranded oligonucleotides as recognition probes for the reagentless, single-step, electrochemical detection of anti-DNA antibodies directly in blood serum.
Asunto(s)
Anticuerpos/sangre , Anticuerpos/inmunología , Análisis Químico de la Sangre/métodos , ADN/inmunología , Animales , Anticuerpos/química , Bovinos , ADN/química , Sondas de ADN/química , Sondas de ADN/inmunología , Electroquímica , Electrodos , Oro/química , Inmunoensayo , Límite de Detección , Modelos Moleculares , Conformación de Ácido Nucleico , Sistemas de Atención de Punto , Conformación Proteica , Factores de TiempoAsunto(s)
Fosfatasa Alcalina , Sondas de ADN/síntesis química , Compuestos Heterocíclicos , Biología Molecular/métodos , Anticuerpos/genética , Sondas de ADN/inmunología , Sondas de ADN/aislamiento & purificación , Fluoresceína , Fluoresceínas , Haptenos/genética , Compuestos Heterocíclicos con 1 Anillo , Indicadores y Reactivos , Mediciones Luminiscentes , Hibridación de Ácido NucleicoAsunto(s)
Biotina , Digoxigenina , Hibridación in Situ/métodos , Animales , Anticuerpos/genética , Sondas de ADN/genética , Sondas de ADN/inmunología , Sondas de Oligonucleótidos/genética , Sondas de Oligonucleótidos/inmunología , Hipófisis/citología , Sondas ARN/genética , Sondas ARN/inmunología , RatasAsunto(s)
Complemento C4a/biosíntesis , Sondas de ADN/inmunología , Antígenos HLA-B/biosíntesis , Antígenos HLA-DR/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Linfocitos T/inmunología , Trasplante Heterólogo/inmunología , Animales , Linfocitos B/inmunología , Southern Blotting , Perros , Rechazo de Injerto/inmunología , Cadenas HLA-DRB1 , Prueba de Histocompatibilidad , Humanos , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Complejo Mayor de Histocompatibilidad , Porcinos , Porcinos EnanosRESUMEN
In order to detect the mRNA transcribed from the multidrug-resistance gene (MDR1), thymine-thymine (T-T) dimerized single-stranded DNA probes have been utilized for hybridization with mRNA either on nitrocellulose filters or in cells and tissues. S1 nuclease digestion rather than sonication was used to obtain short T-T dimerized single-stranded DNA (300-400 bases) so that they could penetrate well into the cytoplasm. The hybridized T-T DNA was detected immunohistochemically using rabbit anti-T-T DNA antibody (Ab) and peroxidase-labeled goat anti-rabbit IgG Ab. Employing this system, MDR1 mRNA could be localized clearly in the human multidrug-resistant cell lines K562/ADM, CEM/VLB, 2780AD, and KBC4 cells as well as in human fetal kidney and gastric carcinoma. Furthermore, our system successfully detected the expression of MDR1 mRNA in cell lines of increasing resistance. These results paralleled results obtained at the protein level by immunohistochemistry. The analysis of MDR1 RNA expression by this in situ hybridization technique should be useful in the study of normal human tissues and tumor samples expressing the MDR1 gene.