S. Lusvarghi, J. Sztuba-Solinska, KJ Purzycka, JW Rausch e SFJ Le Grice sono supportate dal programma di ricerca intramurale del National Cancer Institute, National Institutes of Health, USA.

<ol>
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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. 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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. 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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. 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Nessun conflitto di interessi dichiarati.

Presentiamo qui un protocollo dettagliato per la forma ad alta velocità, una tecnica che permette la determinazione della struttura secondaria a risoluzione singolo nucleotide di RNA di qualsiasi dimensione. Inoltre, abbinando i dati di forme sperimentali con secondari algoritmi di predizione di struttura facilita la generazione di modelli di RNA in 2D con un maggior grado di precisione di quanto sia possibile con uno dei due metodi da solo. La combinazione di primer fluorescenza marcata e automatizzato CE offre signif...

Per capire le funzioni di RNA catalitici e non codificanti coinvolti nella regolazione dello splicing, la traduzione, la replicazione del virus e il cancro, una conoscenza dettagliata della struttura dell&#39;RNA è richiesto 1,2. Purtroppo, la previsione accurata di RNA folding rappresenta una sfida formidabile. Agenti di sondaggio classici soffrono molti svantaggi come la tossicità, incompleta copertura dei nucleotidi e / o velocità limitata a 100-150 nucleotidi per esperimento. Senza aiuto secondarie algoritmi di predizione di struttura sono allo stesso modo svantaggioso, a causa di imprecisioni derivanti dalla loro incapacità di distinguere efficacemente tra strutture energeticamente simili. Grandi RNA in particolare sono spesso refrattari ai metodi di determinazione della struttura 3D come cristallografia a raggi X e risonanza magnetica nucleare (NMR), grazie alla loro flessibilità conformazionale e grandi quantità di campioni altamente puri richiesti per queste tecniche. High-capacità SHAPE risolve molti di questi problemi fornendo un efficace, semplice approccio per sondare le strutture di grandi RNA a singolo nucleotide risoluzione. Inoltre, i reagenti utilizzati per la forma sono sicuro, facile da maneggiare e, a differenza della maggior parte dei reagenti chimici probing, reagiscono con tutti e quattro i ribonucleotidi. Questi reagenti possono anche penetrare le membrane cellulari, rendendo possibile sondare RNA nel loro contesto in vivo (s) 3. Originariamente sviluppato nel laboratorio 4 settimane, SHAPE è stato utilizzato per analizzare una grande varietà di RNA, l&#39;esempio più notevole è determinazione della struttura secondaria completa del ~ 9 kb HIV-1 RNA genoma 5. Altri risultati di rilievo utilizzando FORMA includono delucidazione delle strutture di viroidi infettive 6, RNA umani lunghi non codificanti 7, ribosomi lievito 8 e riboswitches 9, nonché per identificare i siti di legame delle proteine ​​nel virione-associata HIV-1 RNA 3. While le varianti originali e high-throughput del protocollo SHAPE sono stati pubblicati altrove 10-12, il presente lavoro fornisce una descrizione dettagliata della determinazione della struttura secondaria dell&#39;RNA da SHAPE high-throughput utilizzando oligonucleotidi fluorescenti, la Beckman Coulter CEQ 8000 Genetic Analyzer, e (v5.3) software SHAPEfinder e RNAStructure. Sono inclusi anche inediti dettagli tecnici e consigli sulla risoluzione dei problemi. Variazioni di forma L&#39;essenza di SHAPE e sue variazioni è l&#39;esposizione di RNA in soluzione acquosa a anidridi elettrofili che acilato selettivamente 2&#39;-idrossile (2&#39;-OH) gruppi ribosio, producendo addotti ingombranti nei siti di modificazione. Questa reazione chimica serve come mezzo di interrogare dinamica strutturale RNA locali, come nucleotidi a singolo filamento sono più inclini ad adottare conformazioni favoriscano attacco elettrofilo da questi reagenti, mentre la base accoppiato o architettonico fabbrnucleotidi ained sono meno o non reattivo 10. Siti di formazione di addotti vengono rilevati mediante trascrizione inversa avviando da fluorescente o radioattivo primer ibridati ad un sito specifico sulla RNA modificato (il &quot;(+)&quot; reazione di innesco di estensione). Quando trascrittasi inversa (RT) non riesce ad attraversare i ribonucleotidi acil derivati, un pool di cDNA prodotti viene prodotto il cui lunghezze coincidono con i siti di modificazione. Un controllo, &quot;(-)&quot; primer estensione reazione utilizzando RNA che non è stato esposto a reagente viene eseguita anche in modo che la chiusura prematura della sintesi del DNA (vale a dire &quot;stop&quot;) a causa di RNA struttura, non specifico filamento di RNA rotture, ecc, maggio. essere distinta da facendo una pausa prodotto mediante modificazione chimica. Infine, due reazioni di sequenziamento dideossi-avvio dagli stessi primer sono utilizzati come marcatori per correlare nucleotidi reattivi con la sequenza primaria di RNA dopo elettroforesi. In applicazione originale di SHAPE, lo stesso 32 P-end-marcato primer viene utilizzato per il (+), (-), e due reazioni di sequenziamento. Prodotti di queste reazioni sono caricati in pozzetti adiacenti in un gel di poliacrilammide lastra 5-8%, e frazionato mediante elettroforesi su gel denaturante di poliacrilammide (PAGE; Figura 1). Analisi quantitativa delle immagini gel prodotte da SHAPE convenzionale può essere eseguita utilizzando SAFA, un software semi-automatico di analisi footprinting 13. In contrasto, la forma ad alta velocità impiega primer fluorescente ed elettroforesi capillare automatizzata. Specificamente, per ogni regione di RNA in esame, una serie di quattro primer DNA avente una sequenza comune ma diverso 5 &#39;etichette fluorescenti devono essere sintetizzati o acquistati. Questi oligonucleotidi diversamente etichettati servono a primi due reazioni forma e le due reazioni di sequenziamento, i cui prodotti sono raggruppati e frazionato / rilevati mediante elettroforesi capillare automatizzato (CE). Whereas il profilo di reattività 100-150 nt di RNA può essere ottenuto da una serie di quattro reazioni usando l&#39;approccio originale, SHAPE alta produttività consente risoluzione del 300-600 nt da un singolo campione riunito 3. Fino a 8 serie di reazioni può essere frazionata simultaneamente, mentre ben 96 campioni possono preparato per frazionamento nel corso di 12 consecutivi piste CE (Figura 2). Inoltre, il software SHAPEfinder, sviluppato per elaborare e analizzare i dati che emergono dal QVC e altri analizzatori genetici, è più automatizzato e richiede meno interventi da parte dell&#39;utente di SAFA 13 o altri pacchetti di gel di analisi. Metodologie high-throughput più avanzati hanno recentemente emerso come PARS (analisi in parallelo di struttura RNA) 14 e Frag-Seq (frammento-sequencing) 15, che utilizzano enzimi struttura-specifiche, piuttosto che reattivi alchilazione in combinazione con tecniche di sequenziamento di nuova generazione per ottenere information su la struttura dell&#39;RNA. Nonostante l&#39;attrattiva di queste tecniche, le molte limitazioni inerenti alla nucleasi tastatura rimangono ancora 16. Questi problemi possono essere aggirate nel sequenziamento SHAPE (SHAPE-Seq) protocollo 17, dove sequenziamento di nuova generazione è preceduta dalla modificazione chimica e trascrizione inversa di RNA in una maniera simile a quella effettuata per SHAPE convenzionale. Mentre questi metodi possono rappresentare il futuro della determinazione della struttura dell&#39;RNA, è importante ricordare che sequenziamento di nuova generazione è molto costoso, e rimane non disponibile per molti laboratori. FORMA Data Analysis Dati prodotti nell&#39;analizzatore genetico è presentato nella forma di un elettroferogramma, in cui l&#39;intensità di fluorescenza del campione (s) che fluisce attraverso il rivelatore capillare è tracciata contro un indice di tempo di migrazione. Questa trama prende la forma di tracce sovrapposte corrispondenti ai quattro canali di fluorescenzas utilizzato per rilevare i diversi fluorofori, e dove ogni traccia è composta da picchi corrispondenti ai singoli prodotti di cDNA o sequenziamento. Dati elettroferogramma vengono esportati dalla analizzatore genetico come file di testo delimitato da tabulazioni e importati in trasformazione ShapeFinder e software di analisi 18. ShapeFinder è inizialmente utilizzato per eseguire una serie di trasformazioni matematiche sui dati per garantire che i tempi di migrazione e di volumi di picco rispecchiano fedelmente le identità e le quantità dei prodotti di reazione, rispettivamente. Picchi sono poi allineati e integrati, ed i risultati tabulati insieme alla sequenza di RNA primario. Un &quot;profilo reattività&quot; per il segmento pertinente del RNA è ottenuto sottraendo i valori di controllo della (+) valori associati a ogni RNA nucleotide, e normalizzare i dati come descritto di seguito. Questo profilo viene importato in RNAstructure (v5.3) 19,20 software che converte la reattività val normalizzatoUES in vincoli pseudo-energia che sono incorporati nella algoritmo pieghevole struttura secondaria dell&#39;RNA. Combinando chemical probing e piegatura algoritmi in questo modo migliora significativamente l&#39;accuratezza di previsione strutturale rispetto a uno metodo da solo 12,21. L&#39;uscita di RNAstructure (v5.3) comprende le immagini di più bassa energia RNA strutture secondarie con codice colore Con il profilo SHAPE reattività (s), così come le stesse strutture in testuale notazione dot-staffa. Questi ultimi possono essere successivamente esportati in software dedicato alla visualizzazione grafica di RNA struttura secondaria come Varna 22 e PseudoViewer 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. Diagramma di flusso della determinazione della struttura dell&#39;RNA tramite SHAPE 4,10. (A) RNA may essere ottenuto da campioni biologici o di trascrizione in vitro. (B) A seconda della sorgente, l&#39;RNA è piegato o altrimenti elaborati e modificati con il reattivo di SHAPE. (C) trascrizione inversa utilizzando fluorescente o radioattiva primer marcati. (d) i prodotti di cDNA sono frazionato sia tramite elettroforesi su gel a base capillare o lastra. (E) Analisi Fragment. (F) previsione della struttura dell&#39;RNA. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura.</a> <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> Figura 2. Il carattere high-throughput di SHAPE CE-based permette una rapida analisi di molteplici RNA, e / o più segmenti dello stesso RNA. (A) </strong&gt; Rappresenta come un RNA può essere diviso in sezioni 300-600 nt (codice colore in verde, blu e rosso) (B) Sezioni di RNA vengono sondati in modo indipendente utilizzando diversi set di primer fluorescenti (frecce nere) (C) imposta di Le reazioni sono raggruppati e caricati nei pozzetti A1, B1, C1, ecc, rispettivamente, fornisce una copertura completa per il ~ 3 kb RNA1. Prodotti di reazione di RNA 2, 3, 4, ecc possono essere preparati in modo simile per frazionamento in corse elettroforetiche consecutivi. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">Clicca qui per ingrandire 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

Comprendere la funzione di RNA coinvolti in processi biologici richiede una conoscenza approfondita della struttura dell&#39;RNA. A questo fine, la metodologia denominata &quot;2 selettivo high-throughput &#39;acilazione idrossile analizzato da primer extension&quot;, o la forma, permette la previsione di RNA struttura secondaria con risoluzione singolo nucleotide. Questo approccio utilizza agenti chimici che probing acilato preferenzialmente singole regioni o flessibile filamento di RNA in soluzione acquosa. Siti di modificazione chimica vengono rilevati mediante trascrizione inversa del RNA modificati, ed i prodotti di questa reazione sono frazionati mediante elettroforesi capillare automatizzato (CE). Dal momento che la transcriptasi inversa si ferma a quei nucleotidi di RNA modificati dai reagenti SHAPE, la libreria di cDNA risultante indirettamente associa quei ribonucleotidi che sono a singolo filamento nel contesto delle RNA piegati. Utilizzando il software ShapeFinder, i elettroferogrammi prodotti dalla automatizzato CE vengono elaborati e convertiti in nutavoli reattività cleotide che sono a loro volta convertiti in vincoli pseudo-energetiche utilizzate nel RNAStructure (V5.3) algoritmo di predizione. Le strutture di RNA bidimensionali ottenute combinando SHAPE sondando con in silico RNA predizione della struttura secondaria sono stati trovati per essere molto più preciso di strutture ottenute utilizzando il metodo da solo.

RNA contenente il virus HIV-1 rev risposta elemento (RRE) ea 3 &#39;terminale cassetta struttura 4 è stato preparato da un plasmide linearizzato mediante trascrizione in vitro, dopo di che è stato piegato da riscaldamento, raffreddamento e incubazione a 37 ° C in presenza di MgCl 2. RNA è stato esposto a NMIA e poi invertire trascritto da un primer di DNA 5&#39;-end-marcato ibridato a cassetta struttura terminale 3 &#39;. La libreria di cDNA risultante SHAPE, insieme a reazioni di cont...

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

High-throughput selettivo 2 &#39;acilazione idrossile analizzato da primer extension (SHAPE) utilizza un romanzo chemical probing tecnologia, la trascrizione inversa, elettroforesi capillare e software di predizione della struttura secondaria per determinare le strutture di RNA da diverse centinaia a diverse migliaia di nucleotidi a singolo nucleotide risoluzione.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

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

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Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

31.2K Views.  Frederick National Laboratory for Cancer Research. High-throughput selettivo 2 'acilazione idrossile analizzato da primer extension (SHAPE) utilizza un romanzo chemical probing tecnologia, la trascrizione inversa, elettroforesi capillare e software di predizione della struttura secondaria per determinare le strutture di RNA da diverse centinaia a diverse migliaia di nucleotidi a singolo nucleotide risoluzione.

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.

High-throughput iperespressione schermo lievito plasmidi

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

Ricerca

Biologia

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 protocollo per la Computer-Based Protein Structure Prediction e Funzione

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.

Cristallizzazione high-throughput di proteine ​​di membrana con il metodo lipidica 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.

High-throughput mappatura fisica dei cromosomi con modalità automatizzate<em&gt; In situ</em&gt; Ibridazione

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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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 manuale Schermo piccole molecole si avvicina ad alta velocità Uso embrioni di zebrafish

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.

Legatura altamente efficiente delle piccole molecole di RNA per microRNA Quantificazione mediante sequenziamento High-Throughput

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

Rivelazione di bersagli miRNA in high-throughput usando il saggio 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.

Identificazione delle coppie Kinase-substrato Usando 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 ...