Name: monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting
Uploaded: 2011-10-17T13:00:00
Description: Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Diese Arbeit wurde durch Zuschüsse aus dem National Institute of Health RO1-GM085130 und National Science Foundation MCB0929394 unterstützt. Wir danken Dr. Marion Schmidt für ihre Gastfreundschaft und für die Erlaubnis zu filmen in ihrem Labor.

1. Vorbereitung der Reagenzien Footprinting <ol><li> Bereiten Sie einen 10x Reaktionspuffer mit 100 mM Natriumcacodylat, 1 mM EDTA und 1 M KCl. Den pH-Wert auf 7,4. Filtern Sie die Puffer unter Verwendung eines 0,2 uM Acetat-Filter-Gerät (Nalgene). Bemerkung: nicht pipet RNA direkt in 10x Puffer. </li><li> Bereiten Sie die Titrationsreaktion Mix für jede Reaktion, wie in Tabelle 1 angegeben. Das Volumen der Titration Mix (1x Puffer und Mg (II) in der gewünschten Konzentration) sollte 90 ul werden, bevor man 10 &amp...

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Hydroxyl-Radikal Footprinting ist ein wertvolles Instrument, um das Lösungsmittel zugängliche Oberfläche von Nukleinsäuren zu beurteilen. Qualitative und quantitative Bildung der Tertiärstruktur 14 kann als eine Funktion von Parametern wie Ionen-Art und Konzentration, pH, Temperatur, bindende Proteine ​​oder Falten Co-Faktoren zu beachten. Die überzeugende Kombination aus einem geradlinig und kostengünstige Protokoll und die daraus resultierende Lösungsmittel Zugänglichkeit und Falten Information...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA-Moleküle spielen eine wesentliche Rolle in der Biologie. Neben der Übertragung genetischer Information, kann RNA in einzigartige Tertiärstrukturen Erfüllung einer bestimmten biologischen Rolle als Regulierer, Bindemittel oder Katalysator zu falten. Informationen über tertiäre Bildung Kontakt ist unerlässlich, um die Funktion von RNA-Molekülen zu verstehen. Hydroxyl-Radikale (• OH) sind einzigartig Sonden der Struktur von Nukleinsäuren aufgrund ihrer hohen Reaktivität und geringen Größe. 1 Wenn ein footprinting Sonde verwendet, map Hydroxylradikale das Lösungsmittel zugängliche Oberfläche des Phosphodiester-Rückgrat der DNA 1 und RNA 2 mit so fein wie single nucleotide Auflösung. Hydroxyl-Radikal footprinting können die Nukleotide innerhalb einer intermolekularen Kontaktfläche, in DNA-Protein-1 und RNA-Protein-Komplexe z. B. zu identifizieren. Equilibrium 3 und 4 kinetische Übergänge können durch die Durchführung Hydroxylradikal Footprinting als Funktion einer Lö bestimmt werdenauf variable oder Zeit, bzw.. Ein wesentliches Merkmal des Fußabdrucks ist, dass eine begrenzte Exposition mit der Sonde (zB &quot;Single-Hit-Kinetik&quot;) führt in die einheitliche Probenahme jedes Nukleotid des Polymers. 5 In diesem Video-Artikel verwenden wir die P4-P6-Domäne des Tetrahymena Ribozym RNA Probenvorbereitung und die Bestimmung eines Mg (II)-vermittelte Faltung Isothermen zeigen. Wir beschreiben die Verwendung der bekannten Hydroxyl-Radikal footprinting Protokoll, das H 2 O 2 (wir nennen dies den &quot;peroxidative&quot;-Protokoll) und eine wertvolle, aber nicht allgemein bekannt, dass alternative natürlich gelösten O 2 verwendet (wir nennen so bedarf dies der &#39; oxidative &#39;Protokoll). Eine Übersicht über die Datenreduktion, Transformation und Analyse-Verfahren vorgestellt.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Dieses Protokoll beschreibt, wie die Mg (II)-abhängige Bildung von RNA Tertiärstruktur durch zwei Methoden der Hydroxyl-Radikal footprinting quantifizieren.

Überwachung Equilibrium Veränderungen im RNA-Struktur von &quot;peroxidativer&quot; und &quot;Oxidativer &#39;Hydroxylradikal Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

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