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

Detta arbete har finansierats med bidrag från National Institute of Health RO1-GM085130 och National Science Foundation MCB0929394. Vi tackar Dr Marion Schmidt för sin gästfrihet och för att tillåta oss att filma i hennes laboratorium.

1. Beredning av footprints reagenser <ol><li> Förbered en 10x reaktion buffert som innehåller 100 cacodylate mM natriumklorid, 1 mM EDTA, och 1 M KCl. Justera pH till 7,4. Filtrera bufferten med hjälp av en 0,2 mikroM enhet acetat filter (Nalgene). Anmärkning: inte Pipettera RNA direkt till 10x buffert. </li><li> Förbered mixen titrering reaktion för varje reaktion som anges i tabell 1. Volymen av titreringen mix (1x buffert och Mg (II) på önskad koncentration) bör vara 90 l, innan du lägger 10μl av RNA i 1...

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Hydroxylradikaler footprints är ett värdefullt verktyg för att bedöma lösningsmedlet åtkomlig yta av nukleinsyror. Kvalitativa och kvantitativa bildandet av tertiärstruktur 14 kan följas som en funktion av parametrar som jon-typ och koncentration, pH, temperatur, bindande proteiner eller vikning co-faktorer. Den övertygande kombination av en rak och billig protokoll och den resulterande lösningsmedel tillgänglighet och vikning information på en enda nukleotid nivå gör denna metod mycket attrakti...

<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-molekyler spelar en viktig roll i biologi. Förutom att överföra genetisk information, kan RNA lägga till unika tertiära strukturer som uppfyller en viss biologisk roll som regulator, bindemedel eller katalysator. Information om tertiära kontakt bildning är avgörande för att förstå funktionen av RNA-molekyler. Hydroxylradikaler (• OH) är unika sonder av strukturen hos nukleinsyror grund av sin höga reaktivitet och liten storlek. 1 När den används som en footprints sond, hydroxylradikaler karta lösningsmedlet åtkomlig yta phosphodiester ryggraden i DNA-1 och RNA 2 med lika bra som enda nukleotid upplösning. Hydroxylradikaler footprints kan användas för att identifiera nukleotider i en intermolekylär kontaktyta, t ex i DNA-protein-1 och RNA-protein komplex. Jämvikt 3 och kinetiska 4 övergångar kan bestämmas genom att utföra hydroxylradikaler footprints som en funktion av en solutipå rörliga eller tid, respektive. En viktig funktion i footprints är att begränsad exponering till sonden (t.ex. &quot;single-hit kinetik&quot;) resulterar i en enhetlig provtagning av varje nukleotid av polymer. 5 I den här videon artikeln använder vi den P4-P6 domän Tetrahymena ribozyme att illustrera RNA provberedning och bestämning av ett Mg (II)-medierad fällbara isotermer. Vi beskriver användningen av den välkända hydroxylradikaler footprints protokoll som kräver H 2 O 2 (Vi kallar detta &quot;peroxidative&quot; protocol) och en värdefull, men inte allmänt känt, alternativ som använder naturligt löst O 2 (Vi kallar detta &quot; oxidativ &quot;Protocol). En översikt av datareduktion, omvandling och förfaranden analys presenteras.

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

Detta protokoll beskriver hur man kvantifiera Mg (II)-beroende bildning av RNA-tertiär struktur genom två metoder för hydroxylradikaler footprints.

Övervakning Jämvikt Förändringar i RNA-struktur med &quot;Peroxidative&quot; och &quot;oxidativ&quot; hydroxylradikaler footprints

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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