S. Lusvarghi, J. Sztuba-Solinska, KJ Purzycka, JW Rausch und SFJ Le Grice werden durch die Interne Research Program des National Cancer Institute, National Institutes of Health, USA unterstützt.

<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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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. 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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. 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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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Wir stellen Ihnen hier eine detaillierte Protokoll für High-Throughput-SHAPE, eine Technik, die sekundäre Struktur Bestimmung erlaubt single-nucleotide Auflösung für RNAs jeder Größe. Außerdem Kopplung experimentellen Daten mit SHAPE Sekundärstruktur Vorhersage-Algorithmen ermöglicht Generation von RNA 2D-Modelle mit einem höheren Grad an Genauigkeit, als es mit beiden Methoden allein. Die Kombination von fluoreszenzmarkierten Primern und automatisierte CE bietet erhebliche Vorteile gegenüber dem herkömmlich...

Um die Funktionen der katalytischen und nicht-kodierende RNAs bei der Regulation der Spleißen, Übersetzung, Virusreplikation und Krebs beteiligt zu verstehen, ist eine genaue Kenntnis der RNA-Struktur 1,2 erforderlich. Leider stellt genaue Vorhersage der RNA-Faltung eine gewaltige Herausforderung. Klassische Sondieren Mittel leiden viele Nachteile, wie Toxizität, unvollständige Nucleotidsequenz Abdeckung und / oder den Durchsatz beschränkt auf 100-150 Nukleotide pro Versuch. Unaided Sekundärstruktur Vorhersage-Algorithmen sind ähnlich nachteilig wegen Ungenauigkeiten aufgrund ihrer Unfähigkeit, effektiv unter energetisch ähnlichen Strukturen zu unterscheiden. Große RNAs insbesondere auch oft feuerfesten Verfahren zur 3D-Struktur Bestimmung wie Röntgenkristallographie und NMR-Spektroskopie (NMR), die aufgrund ihrer konformativen Flexibilität und große Mengen an hochreinem Proben für diese Techniken erforderlich. HIGH-Durchsatz SHAPE löst viele dieser Probleme durch eine effektive, einfache Methode, um die Erforschung der Strukturen von großen RNAs bei single-nucleotide Auflösung. Darüber hinaus sind die Reagenzien für SHAPE verwendet sichere, einfach zu handhaben und im Gegensatz zu den meisten anderen chemischen Reagenzien Sondieren, mit allen vier Ribonukleotiden reagieren. Diese Reagenzien können auch zelluläre Membranen eindringen, was es ermöglicht, RNAs in ihre in vivo-Kontext (e) 3 sondieren. Ursprünglich in den Wochen 4 Labor entwickelt wurde, hat SHAPE verwendet worden, um eine Vielzahl von RNAs, die bemerkenswerteste Beispiel ist die Bestimmung der vollständigen sekundären Struktur des ~ 9 kb HIV-1-RNA-Genom 5 zu analysieren. Andere bemerkenswerte Erfolge mit SHAPE umfassen Aufklärung der Strukturen von infektiösen Viroiden 6, menschlichen langen nicht-kodierenden RNAs 7, Hefe Ribosomen 8 und 9 riboswitches sowie zur Identifizierung von Protein-Bindungsstellen in Virion-assoziierten HIV-1-RNA-3. While das Original und High-Throughput-Variationen des SHAPE-Protokoll wurde bereits an anderer Stelle veröffentlicht 10-12, stellt die vorliegende Arbeit eine detaillierte Beschreibung der RNA Sekundärstruktur Bestimmung durch High-Throughput-SHAPE mit fluoreszierenden Oligonukleotiden, die Beckman Coulter CEQ 8000 Genetic Analyzer, und ShapeFinder und RNAStructure (v5.3)-Software. Bisher unveröffentlichte technischen Details und Hinweise zur Fehlersuche sind ebenfalls enthalten. Variationen von SHAPE Das Wesen Form und seinen Varianten ist die Exposition von RNA in wässriger Lösung einer elektrophilen Anhydride, die selektiv acyliert 2&#39;-Hydroxy-(2&#39;-OH)-Gruppen Ribose, wodurch sperrige Addukte an den Stellen der Modifikation. Diese chemische Reaktion dient als Mittel zum Abfragen lokalen RNA strukturelle Dynamik, als einzelsträngige Nukleotide anfälliger für Konformationen förderlich elektrophilen Angriff durch diese Reagenzien erlassen sind, während basengepaarte oder architektonisch bebauteained Nukleotide sind weniger reaktiv oder 10. Seiten der Adduktbildung werden durch reverse Transkription initiiert von fluoreszent oder radioaktiv markierten Primer hybridisiert an eine spezifische Stelle auf der modifizierten RNA (die &quot;(+)&quot; Primer-Extension-Reaktion) detektiert. Als reverse Transkriptase (RT), um die acylierten Ribonukleotide durchqueren fehlschlägt, wird ein Pool von cDNA-Produkte hergestellt werden, deren Längen übereinstimmen mit Seiten der Modifikation. Ein Steuersystem, &quot;(-)&quot; Primerverlängerungsreaktion Verwendung RNA, die nicht Reagenz ausgesetzt wurde auch durchgeführt, so dass eine vorzeitige Beendigung der DNA-Synthese (dh &quot;Haltestellen&quot;) aufgrund von Struktur, unspezifische RNA-Strang Bruch etc, kann RNA. Pausieren von durch chemische Modifikation hergestellt wurden, unterschieden werden. Schließlich werden zwei Dideoxy-Sequenzierungsreaktionen Einleiten von denselben Primern als Marker verwendet werden, um reaktive Nukleotide mit dem RNA-Primärsequenz nach Elektrophorese korrelieren. In der ursprünglichen Anwendung SHAP(-), Und zwei Sequenzierungsreaktionen E, gleich 32 P-end-markiertem Primer für die (+), verwendet. Produkte dieser Reaktionen in benachbarten Vertiefungen in einer 5-8% igen Polyacrylamid-Slab-Gel geladen und fraktioniert durch denaturierende Polyacrylamid-Gelelektrophorese (PAGE; Abbildung 1). Die quantitative Analyse der Gel-Bilder durch herkömmliche Form hergestellt werden unter Verwendung SAFA, ein halbautomatisches Footprint-Analyse-Software 13 ist. Im Gegensatz dazu verwendet Hochdurchsatz-FORM fluoreszenzmarkierten Primer und automatisierten Kapillarelektrophorese. Genauer gesagt, für jede Region von RNA untersucht, ein Satz von vier DNA Primer eine gemeinsame Sequenz, aber unterschiedliche 5&#39;Fluoreszenzmarkierungen müssen synthetisiert oder gekauft werden. Diese unterschiedlich markierten Oligonukleotide dienen prime zwei SHAPE Reaktionen und zwei Sequenzierungsreaktionen, deren Produkte gebündelt und fraktionierte / erkannt durch automatisierte Kapillarelektrophorese (CE). Whereas die Reaktivität Profil von 100-150 nt von RNA aus einem Satz von vier Reaktionen mit dem ursprünglichen Ansatz erreicht werden kann, ermöglicht Hochdurchsatz-SHAPE Auflösung von 300-600 nt aus einer gepoolten Probe 3. Bis zu 8 Sätze von Reaktionen fraktioniert werden gleichzeitig, jedoch nicht weniger als 96 Proben für die Fraktionierung innerhalb von 12 aufeinander folgenden Läufen CE (2) hergestellt. Darüber hinaus ist die ShapeFinder entwickelte Software zur Verarbeitung und Analyse von Daten, die aus dem CEQ und andere genetische Analysatoren, stärker automatisiert und erfordern viel weniger Benutzereingriff als SAFA 13 oder Gel-Analyse-Pakete. Weiter fortgeschrittene Hochdurchsatz-Methodik wurden kürzlich wie PARS (parallele Analyse von RNA-Struktur) 14 und Frag-Seq (Fragment-Sequenzierung) 15, die Struktur-spezifische Enzyme anstatt Alkylierungsmittel Verwendung in Verbindung mit der nächsten Generation Sequenzierung Techniken zu erhalten entstanden information über RNA-Struktur. Trotz der Attraktivität dieser Techniken, die viele Einschränkungen inhärent Nuklease Sondieren noch 16 bleiben. Diese Probleme können in Form Sequenzierung (Shape-Seq) 17-Protokoll, wobei Sequenzierung der nächsten Generation durch chemische Modifizierung und reverse Transkription der RNA in einer ähnlichen Weise wie in herkömmliche Form durchgeführt vorausgeht umgangen werden. Obwohl diese Methoden die Zukunft der RNA-Struktur Bestimmung darstellen kann, ist es wichtig, sich daran zu erinnern, dass der nächsten Generation Sequenzierung sehr teuer ist und bleibt für viele Labore. SHAPE Data Analysis Die Daten in der Genetic Analyzer erzeugt wird in Form einer Elektropherogramm dargestellt, wobei die Fluoreszenzintensität der Probe (n), der durch die Kapillare Detektor wird gegen einen anderen Zeitpunkt aufgetragen. Dieses Grundstück hat die Form von überlappenden Spuren entsprechend den vier Fluoreszenzkanals verwendet, um die verschiedenen Fluorophoren zu erfassen, und wobei jede Spur aus einzelnen Peaks, die cDNA oder Sequenzierungsprodukte besteht. Elektropherogramm Daten aus dem Genetic Analyzer als Tab-getrennte Textdatei exportiert und importiert in ShapeFinder Transformation und Analyse-Software 18. ShapeFinder zunächst verwendet, um eine Reihe mathematischer Transformationen an den Daten durchführen, um sicherzustellen, dass die Migration Zeiten und Peakvolumina genau die Identität und Menge der Reaktionsprodukte sind. Peaks werden dann ausgerichtet und integriert, und die Ergebnisse zusammen mit dem primären RNA-Sequenz aufgelistet. A &quot;Reaktivitätsprofil&quot; für den betreffenden Abschnitt des RNA wird durch Subtrahieren Steuerwerte aus dem (+) RNA, die mit jedem Nukleotid verbunden ist, und das Normalisieren der Daten, wie unten beschrieben erhalten. Dieses Profil wird in RNAstructure importiert (v5.3)-Software 19,20, die den normierten Reaktivität val wandeltUES in Pseudo-Energie Einschränkungen, die in der RNA-Sekundärstruktur Faltungsalgorithmus eingebaut sind. Das Kombinieren der chemischen Sondieren und Falten Algorithmen auf diese Weise erheblich verbessert die Genauigkeit der Vorhersage Struktur im Vergleich zu beiden Verfahren allein 12,21. Der Ausgang des RNAstructure (v5.3) enthält Bilder von der niedrigsten Energie RNA Sekundärstrukturen mit der Form Reaktivitätsprofil (en), sowie den gleichen Strukturen in textueller dot-Klammern zu farbcodiert. Letzteres kann anschließend auf Software spezialisiert auf die grafische Darstellung der RNA Sekundärstruktur wie Varna 22 und 23 PseudoViewer exportiert werden. <img alt="Abbildung 1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> Abbildung 1. Flussdiagramm der RNA-Struktur Bestimmung über SHAPE 4,10. (A) RNA may aus biologischen Proben oder durch in vitro-Transkription erhalten werden. (B) Je nach Quelle wird RNA gefaltet oder anderweitig verarbeitet und modifiziert mit SHAPE Reagenz. (C) reverse Transkription fluoreszierend oder radioaktiv markierte Primer verwenden. (D) cDNA-Produkte sind fraktionierten entweder über Kapillare oder Slab-Gel-basierten Elektrophorese. (E) Fragment-Analyse. (F) RNA-Struktur-Vorhersage. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">Klicken Sie hier für eine größere Abbildung zu sehen.</a> <img alt="Abbildung 2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> Abbildung 2. Die Hochdurchsatz-Charakter der CE-Form ermöglicht eine schnelle Analyse mehrerer RNAs, und / oder mehrere Segmente der gleichen RNA. (A) </stRong&gt; Gibt an, wie ein RNA in 300-600 nt Abschnitte (Farbe in grün, blau und rot kodiert) (B) Sektionen der RNA unabhängig sondiert mit verschiedenen Sätzen von fluoreszierenden Primer (schwarze Pfeile) (C) Sets unterteilt werden kann Reaktionen werden vereinigt und in die Vertiefungen A1, B1, C1, etc., die jeweils geladen, wodurch eine vollständige Abdeckung des ~ 3 kb RNA1. Reaktionsprodukte von RNAs 2, 3, 4, usw. können in ähnlicher Weise für die Fraktionierung in aufeinanderfolgenden Läufen Elektrophorese vorbereitet werden. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">Klicken Sie hier, um eine größere Abbildung anzuzeigen.</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

Das Verständnis der Funktion der RNA in biologischen Prozessen beteiligt erfordert eine gründliche Kenntnis der RNA-Struktur. Zu diesem Zweck wird die Methodik genannt &quot;High-Throughput-selektive 2-Hydroxyl Acylierung durch Primer-Extension analysiert&quot;, oder SHAPE ermöglicht Vorhersage von RNA Sekundärstruktur mit Einzel-Nukleotid-Auflösung. Dieser Ansatz nutzt chemische Sondierung Mittel, die Acylierung bevorzugt einzelsträngig oder flexiblen Bereiche der RNA in wässriger Lösung. Seiten der chemischen Modifizierung durch reverse Transkription der RNA modifiziert detektiert und die Produkte dieser Reaktion sind fraktioniert automatisierten Kapillarelektrophorese (CE). Seit Reverse Transkriptase Pausen an diesen RNA Nukleotide durch die Form Reagenzien modifiziert, die resultierende cDNA-Bibliothek indirekt abbildet diese Ribonukleotiden die einzigen im Rahmen der gefalteten RNA gestrandet sind. Mit ShapeFinder Software werden die Elektropherogrammen durch automatisierte CE produziert verarbeitet und in nugonucleotid Reaktivität Tabellen, die sich in Pseudo-Energie-Einschränkungen in der RNAStructure (v5.3) Vorhersage-Algorithmus verwendet wird, umgewandelt. Die zweidimensionalen RNA-Strukturen durch die Kombination FORM Sondieren mit in silico RNA Sekundärstrukturvorhersage erhalten haben, wurde festgestellt, dass viel genauer als Strukturen erhalten mit beiden Methoden allein.

RNA, die die HIV-1-rev-Response-Element (RRE) und eine 3&#39;-terminale Struktur Kassette 4 aus einem linearisierten Plasmid durch in vitro Transkription hergestellt, wonach es durch Erhitzen, Kühlen, und die Inkubation wurde bei 37 ° gefaltet C in Gegenwart MgCl 2. RNA wurde NMIA belichtet und dann von einem 5&#39;-Ende markierten DNA-Primer hybridisiert das 3&#39;-terminale Struktur Kassette revers transkribiert. Die resultierende Form cDNA-Bibliothek, zusammen mit Steuerungs-und Seque...

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

Hochdurchsatz-selektiven 2-Hydroxyl-Acylierung durch Primer-Extension (FORM) analysiert verwendet einen neuartigen chemischen Tastertechnologie, reverse Transkription, Kapillarelektrophorese und Sekundärstrukturvorhersage Software, um die Strukturen der RNAs, die von mehreren hundert bis zu mehreren tausend bestimmen Nukleotide an Einzel-Nukleotid-Auflösung.

RNA Secondary Structure Prediction Mit High-Throughput-SHAPE

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 Aufrufe.  Frederick National Laboratory for Cancer Research. Hochdurchsatz-selektiven 2-Hydroxyl-Acylierung durch Primer-Extension (FORM) analysiert verwendet einen neuartigen chemischen Tastertechnologie, reverse Transkription, Kapillarelektrophorese und Sekundärstrukturvorhersage Software, um die Strukturen der RNAs, die von mehreren hundert bis zu mehreren tausend bestimmen Nukleotide an Einzel-Nukleotid-Auflösung.

Serie: 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 Hefeplasmid Überexpression Bildschirm

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

Forschung

Biologie

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.

Ein Protokoll für die 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 ...

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.

High-Throughput-Kristallisation von Membranproteinen mit dem Lipidische Bicellen Methode

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-Physikalische Kartierung von Chromosomen unter Verwendung von Automated<em&gt; In-situ-</em&gt; Hybridisierung

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.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

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.

Ein Handbuch Kleine Molecule Bildschirm Nähern Hochdurch Mit Zebrafischembryonen

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.

Hoch effiziente Ligation kleine RNA-Moleküle für MicroRNA Quantifizierung von Hochdurchsatz-Sequenzierung

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.

Hochdurchsatz-Screening für Chemische Modulatoren der Post-transkriptionell regulierten Genen

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.

Nachweis von miRNA Targets in Hochdurchsatz-Verwendung des 3&#39;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 ...

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.

Identifikation von Kinase-Substrat-Pairs Mit 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 ...