S. Lusvarghi, J. Sztuba-Solinska, KJ Purzycka, JW Rausch y SFJ Le Grice son apoyados por el Programa de Investigación Intramural del Instituto Nacional del Cáncer, de los Institutos Nacionales de Salud, EE.UU..

<ol>
	<li>Scott, W. G., Martick, M., Chi, Y. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+regulatory+RNA+elements:+ribozymes+that+regulate+gene+expression.">Structure and function of regulatory RNA elements: ribozymes that regulate gene expression.</a> Biochim. Biophys. Acta. 1789, 634-641 (2009).</li><li>Moore, P. B., Steitz, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+roles+of+RNA+in+the+synthesis+of+protein.">The roles of RNA in the synthesis of protein.</a> Cold Spring Harb. Perspect. Biol. 3, a003780 (2011).</li><li>Wilkinson, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+SHAPE+analysis+reveals+structures+in+HIV-1+genomic+RNA+strongly+conserved+across+distinct+biological+states.">High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states.</a> Plos Biol. 6, 883-899 (2008).</li><li>Merino, E. J., Wilkinson, K. A., Coughlan, J. L., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+structure+analysis+at+single+nucleotide+resolution+by+selective+2+'-hydroxyl+acylation+and+primer+extension+(SHAPE).">RNA structure analysis at single nucleotide resolution by selective 2 '-hydroxyl acylation and primer extension (SHAPE).</a> J. Am. Chem. Soc. 127, 4223-4231 (2005).</li><li>Watts, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+secondary+structure+of+an+entire+HIV-1+RNA+genome.">Architecture and secondary structure of an entire HIV-1 RNA genome.</a> Nature. 460, 711-716 (2009).</li><li>Xu, W., Bolduc, F., Hong, N., Perreault, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+a+combination+of+computer-assisted+structure+prediction+and+SHAPE+probing+to+elucidate+the+secondary+structures+of+five+viroids.">The use of a combination of computer-assisted structure prediction and SHAPE probing to elucidate the secondary structures of five viroids.</a> Mol. Plant Pathol. , (2012).</li><li>Novikova, I. V., Hennelly, S. P., Sanbonmatsu, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+architecture+of+the+human+long+non-coding+RNA,+steroid+receptor+RNA+activator.">Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.</a> Nucleic Acids Res. 40, 5034-5051 (2012).</li><li>Leshin, J. A., Heselpoth, R., Belew, A. T., Dinman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+structural+analysis+of+yeast+ribosomes+using+hSHAPE.">High-throughput structural analysis of yeast ribosomes using hSHAPE.</a> RNA Biol. 8, 478-487 (2011).</li><li>Souliere, M. F., Haller, A., Rieder, R., Micura, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+powerful+approach+for+the+selection+of+2-aminopurine+substitution+sites+to+investigate+RNA+folding.">A powerful approach for the selection of 2-aminopurine substitution sites to investigate RNA folding.</a> J. Am. Chem. Soc. 133, 16161-16167 (2011).</li><li>Wilkinson, K. A., Merino, E. J., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+2+'-hydroxyl+acylation+analyzed+by+primer+extension+(SHAPE):+quantitative+RNA+structure+analysis+at+single+nucleotide+resolution.">Selective 2 '-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution.</a> Nat. Protoc. 1, 1610-1616 (2006).</li><li>McGinnis, J. L., Duncan, C. D. S., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Shape+and+Hydroxyl+Radical+Analysis+of+Rna+Structure+and+Ribonucleoprotein+Assembly.">High-Throughput Shape and Hydroxyl Radical Analysis of Rna Structure and Ribonucleoprotein Assembly.</a> Method Enzymol. 468, 67-89 (2009).</li><li>Low, J. T., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHAPE-directed+RNA+secondary+structure+prediction.">SHAPE-directed RNA secondary structure prediction.</a> Methods. 52, 150-158 (2010).</li><li>Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. S. A. F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-automated+footprinting+analysis+software+for+high-throughput+quantification+of+nucleic+acid+footprinting+experiments.">Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.</a> Rna-a Publication of the Rna Society. 11, 344-354 (2005).</li><li>Kertesz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+measurement+of+RNA+secondary+structure+in+yeast.">Genome-wide measurement of RNA secondary structure in yeast.</a> Nature. 467, 103-107 (2010).</li><li>Underwood, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FragSeq:+transcriptome-wide+RNA+structure+probing+using+high-throughput+sequencing.">FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing.</a> Nat. Methods. 7, 995-1001 (2010).</li><li>Mauger, D. M., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+global+RNA+structure+analysis.">Toward global RNA structure analysis.</a> Nat. Biotechnol. 28, 1178-1179 (2010).</li><li>Lucks, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+RNA+structure+characterization+with+selective+2'-hydroxyl+acylation+analyzed+by+primer+extension+sequencing+(SHAPE-Seq).">Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq).</a> Proc. Natl. Acad. Sci. USA. 108, 11063-11068 (2011).</li><li>Vasa, S. M., Guex, N., Wilkinson, K. A., Weeks, K. M., Giddings, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ShapeFinder:+a+software+system+for+high-throughput+quantitative+analysis+of+nucleic+acid+reactivity+information+resolved+by+capillary+electrophoresis.">ShapeFinder: a software system for high-throughput quantitative analysis of nucleic acid reactivity information resolved by capillary electrophoresis.</a> RNA. 14, 1979-1990 (2008).</li><li>Reuter, J. S., Mathews, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAstructure:+software+for+RNA+secondary+structure+prediction+and+analysis.">RNAstructure: software for RNA secondary structure prediction and analysis.</a> BMC Bioinformatics. 11, 129 (2010).</li><li>Pang, P. S., Elazar, M., Pham, E. A., Glenn, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+RNA+secondary+structure+mapping+by+automation+of+SHAPE+data+analysis.">Simplified RNA secondary structure mapping by automation of SHAPE data analysis.</a> Nucleic Acids Res. 39, e151 (2011).</li><li>Deigan, K. E., Li, T. W., Mathews, D. H., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+SHAPE-directed+RNA+structure+determination.">Accurate SHAPE-directed RNA structure determination.</a> Proc. Natl. Acad. Sci. USA. 106, 97-102 (2009).</li><li>Darty, K., Denise, A., Ponty, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VARNA:+Interactive+drawing+and+editing+of+the+RNA+secondary+structure.">VARNA: Interactive drawing and editing of the RNA secondary structure.</a> Bioinformatics. 25, 1974-1975 (2009).</li><li>Byun, Y., Han, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PseudoViewer:+web+application+and+web+service+for+visualizing+RNA+pseudoknots+and+secondary+structures.">PseudoViewer: web application and web service for visualizing RNA pseudoknots and secondary structures.</a> Nucleic Acids Res. 34, 416-422 (2006).</li><li>Brown, T., Brown, D. J. S., Eckstein, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oligonucleotides and Analogues - A Practical Approach. , 20 (1990).</li><li>Legiewicz, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+Transport+Element+of+the+Murine+musD+Retrotransposon+Requires+Long-range+Intramolecular+Interactions+for+Function.">The RNA Transport Element of the Murine musD Retrotransposon Requires Long-range Intramolecular Interactions for Function.</a> J. Biol. Chem. 285, 42097-42104 (2010).</li><li>Steen, K., Siegfried, N. A., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheis+of+1-methyl-8-nitroisatoic+anhydride+(1M7).">Syntheis of 1-methyl-8-nitroisatoic anhydride (1M7).</a> Protocol Exchange. , (2011).</li><li>Mortimer, S. A., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fast-acting+reagent+for+accurate+analysis+of+RNA+secondary+and+tertiary+structure+by+SHAPE+chemistry.">A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry.</a> J. Am. Chem. Soc. 129, 4144-4145 (2007).</li><li>Mitra, S., Shcherbakova, I. V., Altman, R. B., Brenowitz, M., Laederach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+single-nucleotide+structural+mapping+by+capillary+automated+footprinting+analysis.">High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.</a> Nucleic Acids Res. 36, e63 (2008).</li><li>Giddings, M. C., Severin, J., Westphall, M., Wu, J., Smith, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+software+system+for+data+analysis+in+automated+DNA+sequencing.">A software system for data analysis in automated DNA sequencing.</a> Genome Res. 8, 644-665 (1998).</li><li>Aviran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+and+automation+of+sequencing-based+characterization+of+RNA+structure.">Modeling and automation of sequencing-based characterization of RNA structure.</a> Proc. Natl. Acad. Sci. USA. 108, 11069-11074 (2011).</li></ol>

No hay conflictos de interés declarado.

Se presenta aquí un protocolo detallado para la forma de alto rendimiento, una técnica que permite la determinación de la estructura secundaria de la resolución de un solo nucleótido de ARN de cualquier tamaño. Por otra parte, el acoplamiento de datos experimentales forma con algoritmos de predicción de estructura secundaria facilita la generación de modelos de ARN 2D con un mayor grado de precisión que es posible con cualquiera de los métodos solos. La combinación de cebadores marcados con fluorescencia y au...

Para comprender las funciones de los ARN catalíticos y no codificantes implicadas en la regulación del corte y empalme, traducción, replicación del virus y el cáncer, se requiere un conocimiento detallado de la estructura del ARN 1,2. Desafortunadamente, la predicción exacta de ARN plegado presenta un reto formidable. Agentes de sondeo clásicas sufren de muchas desventajas tales como la toxicidad, la cobertura incompleta de nucleótidos y / o un caudal limitado a 100-150 nucleótidos por experimento. Algoritmos de predicción de estructura secundaria sin ayuda son igualmente desventajoso, debido a las inexactitudes que resultan de su incapacidad para distinguir efectivamente entre estructuras energéticamente similares. Grandes de ARN, en particular, también son a menudo refractario a los métodos de determinación de la estructura 3D, tales como la cristalografía de rayos X y resonancia magnética nuclear (RMN), debido a su flexibilidad conformacional y grandes cantidades de muestras muy puras requeridas para estas técnicas. HFORMA igh rendimiento resuelve muchos de estos problemas al proporcionar un enfoque eficaz, sencilla de sondeo de las grandes estructuras de ARN a una resolución de un solo nucleótido. Por otra parte, los reactivos utilizados para la forma son seguros, fáciles de manejar y, en contraste con la mayoría de otros productos químicos de sondeo reactivos, reaccionan con los cuatro ribonucleótidos. Estos reactivos también pueden penetrar en las membranas celulares, por lo que es posible para sondear RNAs en su contexto in vivo (s) 3. Originalmente desarrollado en las Semanas de laboratorio 4, la forma ha sido utilizado para analizar una amplia variedad de ARN, el ejemplo más notable es la determinación de la estructura secundaria completa del ~ 9 kb VIH-1 ARN del genoma 5. Otros logros notables incluyen SHAPE utilizando elucidación de las estructuras de los viroides infecciosas 6, ARNs no codificantes largas humanos 7, ribosomas de levadura 8 y 9 riboswitches así como para identificar los sitios de unión de proteínas de virión asociada a VIH-1 RNA 3. While las variaciones originales y de alto rendimiento del protocolo SHAPE se han publicado en otros lugares 10 a 12, el presente trabajo ofrece una descripción detallada de la determinación de la estructura secundaria del ARN por la forma de alto rendimiento utilizando oligonucleótidos fluorescentes, la Beckman Coulter CEQ 8000 Genetic Analyzer y software SHAPEfinder y RNAStructure (v5.3). Detalles técnicos inéditas y consejos de solución de problemas también se incluyen. Las variaciones de la forma La esencia de la forma y sus variaciones es la exposición de ARN en solución acuosa a anhídridos electrófilos que acilar selectivamente grupos ribosa 2&#39;-hidroxilo (2&#39;-OH), produciendo aductos voluminosos en los sitios de modificación. Esta reacción química sirve como un medio de interrogar a la dinámica estructural de ARN locales, como los nucleótidos de cadena simple son más propensas a adoptar conformaciones favorables para ataque electrofílico por estos reactivos, mientras que la base emparejado o arquitectónicamente Constrnucleótidos objetivó son menos o no reactivo 10. Los sitios de formación de aductos se detectan mediante transcripción inversa a partir de la iniciación fluorescentemente o radiomarcado cebadores hibridados a un sitio específico en el ARN modificado (el &quot;(+)&quot; reacción de extensión del cebador). Cuando la transcriptasa inversa (RT) no atravesar los ribonucleótidos acilados, un grupo de productos de ADNc se produce cuyas longitudes coinciden con los sitios de modificación. Un control, &quot;(-)&quot; cebador de extensión reacción utilizando ARN que no ha sido expuestos a reactivo también se realiza de modo que la terminación prematura de la síntesis de ADN (es decir, &quot;se detiene&quot;) debido a la estructura del ARN, no específica de ARN de hebra rotura, etc, mayo. distinguirse de deteniéndose producido por modificación química. Por último, dos reacciones de secuenciación didesoxi-inician a partir de los mismos cebadores se utilizan como marcadores para correlacionar nucleótidos reactivos con la secuencia primaria de ARN después de la electroforesis. En la solicitud original de SHAPE, el mismo 32-P marcado en el extremo cebador se utiliza para los (+), -, y dos reacciones de secuenciación (). Los productos de estas reacciones se cargan en pocillos adyacentes en una placa de gel de poliacrilamida al 5-8%, y se fraccionó por electroforesis en gel desnaturalizante de poliacrilamida (PAGE; Figura 1). El análisis cuantitativo de las imágenes de gel producidas por la forma convencional se puede realizar utilizando SAFA, un software de análisis de la huella de semi-automatizado 13. Por el contrario, la forma de alto rendimiento emplea cebadores marcados con fluorescencia y electroforesis capilar automatizado. Específicamente, para cada región de ARN bajo investigación, un conjunto de cuatro cebadores de ADN que tienen una secuencia común pero diferentes 5 &#39;etiquetas fluorescentes deben ser sintetizados o comprados. Estos oligonucleótidos marcados de manera diferente-sirven para dos reacciones principales forma y dos reacciones de secuenciación, los productos de los cuales se agrupan y se fraccionó / ​​detectaron por electroforesis capilar automatizado (CE). WherEAS el perfil de reactividad de los nt 100-150 de ARN se puede obtener a partir de un conjunto de cuatro reacciones utilizando el enfoque original, la forma de alto rendimiento permite la resolución de 300-600 nt a partir de una sola muestra agrupada 3. Hasta 8 conjuntos de reacciones puede ser fraccionado simultáneamente, mientras que tanto como 96 muestras pueden preparado para el fraccionamiento en el transcurso de 12 ejecuciones consecutivas de la CE (Figura 2). Por otra parte, el software SHAPEfinder, desarrollado para procesar y analizar los datos que se desprenden del CEQ y otros analizadores genéticos, es más automatizado y requiere mucho menos intervención del usuario de SAFA 13 u otros paquetes de gel de análisis. Más avanzadas metodologías de alto rendimiento han surgido recientemente tales como PARS (análisis paralelo de la estructura del ARN) 14 y Frag-Seq (fragmento de secuenciación) 15, que utilizan enzimas específicos de la estructura en lugar de reactivos de alquilación en conjunción con técnicas de secuenciación de próxima generación para obtener information sobre la estructura del ARN. A pesar del atractivo de estas técnicas, las muchas limitaciones inherentes a la nucleasa de sondeo siguen siendo 16. Estos problemas pueden ser evitados en la secuenciación FORMA (SHAPE-Seq) 17 protocolo, donde la secuenciación de próxima generación está precedida por modificación química y la transcripción inversa del ARN de una manera similar a la realizada en la forma convencional. Si bien estos métodos pueden representar el futuro de la determinación de la estructura de ARN, es importante recordar que la secuenciación de próxima generación es muy caro, y sigue sin estar disponible para muchos laboratorios. Análisis de datos SHAPE Los datos producidos en el analizador genético se presentan en la forma de un electroferograma, en el que la intensidad de fluorescencia de la muestra (s) que fluye a través del detector capilar se representa en función de un índice de tiempo de migración. Esta parcela tiene la forma de las huellas superpuestas, según el canal cuatro de fluorescencias utiliza para detectar los diferentes fluoróforos, y donde cada traza se compone de los picos correspondientes a los productos de ADNc o secuenciación individuales. Datos electroferograma se exporta desde el analizador genético como un archivo de texto delimitado por tabuladores y se importan en la transformación ShapeFinder y software de análisis 18. ShapeFinder se utiliza inicialmente para llevar a cabo una serie de transformaciones matemáticas en los datos para asegurar que los tiempos de migración y volúmenes pico reflejan con precisión las identidades y cantidades de los productos de reacción, respectivamente. Los picos son entonces alineados e integrados, y los resultados se tabulan junto con la secuencia de ARN primario. Un &quot;perfil de reactividad&quot; para el segmento pertinente de ARN se obtiene restando los valores de control a partir de la (+) valores asociados con cada ARN de nucleótidos, y la normalización de los datos tal como se describe a continuación. Este perfil se importa en RNAstructure (v5.3) Software de 19,20, lo que convierte la val reactividad normalizadaUES en limitaciones pseudo-energía que se incorporan en el algoritmo de plegado estructura secundaria del ARN. La combinación química de sondeo y plegado algoritmos de esta manera mejora significativamente la exactitud de la predicción de la estructura en comparación con cualquiera de los métodos solos 12,21. La salida de RNAstructure (v5.3) incluye imágenes de la energía más baja ARN estructuras secundarias con código de colores con el perfil FORMA reactividad (s), así como las mismas estructuras en Pruebas notación de punto-soporte. Este último puede posteriormente ser exportado al software dedicado a la representación gráfica de la estructura secundaria del ARN tales como Varna PseudoViewer 22 y 23. <img alt="Figura 1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> Figura 1. Organigrama de la estructura de la determinación de ARN a través de SHAPE 4,10. (A) RNA may se obtiene a partir de muestras biológicas o por transcripción in vitro. (B) Dependiendo de la fuente, el ARN se pliega o se procesa y se modificó con el reactivo FORMA lo contrario. (C) La transcripción inversa usando cebadores marcados radiactivamente o fluorescentemente. (D) son productos de ADNc fraccionado ya sea a través de electroforesis capilar a base de gel o de poca altura. (E) Análisis de fragmentos. (F) la predicción de estructura de ARN. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">Haz clic aquí para ver más grande la figura.</a> <img alt="La figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> Figura 2. El carácter de alto rendimiento de FORMA basados ​​en CE permite el análisis rápido de múltiples ARN, y / o múltiples segmentos de los mismos ARN. (A) </strong&gt; Representa cómo una RNA puede ser dividido en secciones 300-600 nt (código de color en verde, azul y rojo) (B) Las secciones del ARN se probaron de forma independiente utilizando diferentes conjuntos de cebadores fluorescentes (flechas negras) (C) juegos de reacciones se combinaron y se cargaron en los pocillos A1, B1, C1, etc, respectivamente, proporcionando una cobertura completa para el ~ 3 kb ARN1. Productos de reacción de RNAs 2, 3, 4, etc pueden prepararse de forma similar para el fraccionamiento de carreras electroforéticas consecutivos. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">Haz clic aquí para ver más grande la figura.</a>

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>N-methylisatoic anhydride (NMIA)</td>
			<td>Life technologies</td>
			<td>M25</td>
			<td>Dissolve in anhydrous DMSO</td>
		</tr>
		<tr>
			<td>1-methyl-t-nitroisatoic anhydride (1M7)</td>
			<td>see ref. 22</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase</td>
			<td>Life technologies</td>
			<td>18080044</td>
			<td>10,000 units</td>
		</tr>
		<tr>
			<td>Thermo sequenase cycle sequencing kit</td>
			<td>Affymetrix</td>
			<td>78500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials provided by the user</td>
		</tr>
		<tr>
			<td>RNA of interest</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6 pmol per reaction (the limit of detection will be determined by the instrument)</td>
		</tr>
		<tr>
			<td>Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.</td>
		</tr>
		<tr>
			<td>DNA template</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers</td>
		</tr>
		<tr>
			<td>10x RNA renaturation buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>5X RNA folding buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)</td>
		</tr>
		<tr>
			<td>2.5X RT mix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4 &mu;l 5X buffer, 1 &mu;l 100 mM DTT, 1.5 &mu;l water,1 &mu;l 10 mM dNTPs, 0.5 &mu;l SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).</td>
		</tr>
		<tr>
			<td>GenomeLab Sample Loading Solution (Beckman Coulter)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Attention: Avoid multiple freeze-thaw cycles</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Capillary electrophoresis</td>
			<td>Beckman</td>
			<td>CEQ8000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>varies</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

rna secondary structure prediction using high-throughput shape

Comprender la función del ARN que participan en los procesos biológicos exige un conocimiento profundo de la estructura del ARN. Con este fin, la metodología denominada &quot;acilación hidroxilo de alto rendimiento 2 selectivo &#39;analizado mediante extensión de cebador&quot;, o forma, permite la predicción de la estructura secundaria de ARN con una sola resolución de nucleótidos. Este enfoque utiliza agentes químicos de sondeo que preferentemente acilato de regiones de cadena sencilla o flexible de ARN en solución acuosa. Sitios de modificación química se detectan mediante transcripción inversa del ARN modificado, y los productos de esta reacción son fraccionados por electroforesis capilar automatizado (CE). Dado que la transcriptasa inversa se detiene en esos nucleótidos de ARN modificados por los reactivos de la forma, la biblioteca de cDNA resultante asigna indirectamente estos ribonucleótidos que son de cadena sencilla en el contexto de los ARN plegadas. El uso de software ShapeFinder, los producidos por electroferogramas automatizado CE son procesados ​​y convertidos en nutablas de reactividad cleotide que se convierten ellos mismos en las limitaciones del pseudo-energía utilizadas en el RNAStructure (v5.3) algoritmo de predicción. Las estructuras de ARN de dos dimensiones obtenidos por combinación de una forma sondeo con ARN in silico de predicción de estructura secundaria se han encontrado para ser mucho más precisa que la estructuras obtenidas usando cualquier método solo.

ARN que contiene el VIH-1 elemento de respuesta rev (RRE) y un 3 &#39;casete estructura de la terminal 4 se preparó a partir de un plásmido linealizado por la transcripción in vitro, después de lo cual se pliega por calentamiento, enfriamiento, y la incubación a 37 ° C en la presencia de MgCl 2. ARN fue expuesto a NMIA y luego a transcripción inversa a partir de un cebador de ADN 5&#39;-marcado en el extremo hibridó con el casete de estructura de la terminal 3 &#39;. La fo...

Watch this Scientific Journal Video about RNA Secondary Structure Prediction Using High-throughput SHAPE at JoVE.com

RNA Secondary Structure Prediction Using High-throughput SHAPE

De alto rendimiento selectiva 2 &#39;acilación hidroxilo analizados por extensión del cebador (FORMA) utiliza un software de predicción de estructura secundaria de producto químico nuevo sondeo de la tecnología, la transcripción inversa, electroforesis capilar y para determinar las estructuras de ARN de varios cientos a varios miles de nucleótidos en una sola resolución de nucleótidos.

ARN de predicción de estructura secundaria Utilizando SHAPE de alto rendimiento

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

rna-secondary-structure-prediction-using-high-throughput-shape

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

31.2K vistas.  Frederick National Laboratory for Cancer Research. De alto rendimiento selectiva 2 'acilación hidroxilo analizados por extensión del cebador (FORMA) utiliza un software de predicción de estructura secundaria de producto químico nuevo sondeo de la tecnología, la transcripción inversa, electroforesis capilar y para determinar las estructuras de ARN de varios cientos a varios miles de nucleótidos en una sola resolución de nucleótidos.

Video: RNA Secondary Structure Prediction Using High-throughput SHAPE

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

La levadura de alto rendimiento de la pantalla sobreexpresión plásmido

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Investigación

Biología

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

Un protocolo para la estructura de la proteína por computadora y la función de predicción

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

De alto rendimiento de cristalización de proteínas de membrana Utilizando el Método lipídico Bicelle

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

De alto rendimiento mapeo físico de cromosomas utilizando automatizado<em&gt; In situ</em&gt; La hibridación

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Selección de alto rendimiento funcional utilizando un doble resplandor de ensayo de luciferasa Homemade

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Ligadura Altamente Eficiente de los Pequeños ARN moléculas de microARN cuantificación mediante secuenciación de alto rendimiento

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Selección de alto rendimiento para moduladores químicos de genes post-transcripcionalmente regulados

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

La detección de los genes miARN objetivos en de alto rendimiento utilizando el ensayo 3&#39;LIFE

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Identificación de los pares quinasa-sustrato Usando selección de alto rendimiento


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...