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

Ce travail a été soutenu par des subventions du National Institute of Health RO1-GM085130 et la National Science Foundation MCB0929394. Nous remercions le Dr Marion Schmidt pour son hospitalité et pour nous permettre de filmer dans son laboratoire.

1. Préparation des réactifs Footprinting <ol><li> Préparer un tampon de réaction 10x contenant 100 mM cacodylate de sodium, EDTA 1 mM, et 1 M KCl. Ajuster le pH à 7,4. Filtrer le tampon en utilisant un dispositif de filtre acétate de 0,2 pM (Nalgene). Remarque: ne pas ARN pipette directement dans la mémoire tampon 10x. </li><li> Préparer le mélange réactif de titrage pour chaque réaction, comme indiqué dans le tableau 1. Le volume du mélange de titration (1x tampon et Mg (II) à la concentration souhaité...

<ol>
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Hydroxyle empreinte radicale est un outil précieux pour évaluer la surface accessible au solvant des acides nucléiques. La formation quantitative et qualitative de la structure tertiaire 14 peut être suivie en fonction de paramètres tels que le type et la concentration d&#39;ions, pH, température, protéines de liaison ou de pliage des co-facteurs. La combinaison convaincante d&#39;un protocole simple et peu coûteux et l&#39;accessibilité résultant de solvant et de l&#39;information pliage sur un seu...

<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

Molécules d&#39;ARN jouent un rôle essentiel dans la biologie. En plus de transmettre l&#39;information génétique, l&#39;ARN peut incorporer uniques structures tertiaires remplir un rôle de régulateur biologiques spécifiques, liant ou de catalyseur. Information sur la formation de contacts tertiaire est essentielle pour comprendre la fonction des molécules d&#39;ARN. Les radicaux hydroxyles (OH •) sont des sondes unique de la structure des acides nucléiques en raison de leur grande réactivité et une petite taille. 1 Lorsqu&#39;il est utilisé comme une sonde d&#39;empreinte, les radicaux hydroxyles cartographier la surface accessible au solvant de l&#39;squelette phosphodiester de l&#39;ADN et l&#39;ARN 1 2 avec aussi fine que la résolution seul nucléotide. Hydroxyle empreinte radical peut être utilisé pour identifier les nucléotides au sein d&#39;une surface de contact intermoléculaires, par exemple en ADN-protéines et d&#39;ARN une teneur en protéines complexes. Equilibre et cinétique 3 4 transitions peut être déterminé en effectuant des empreintes hydroxyles radicaux en fonction d&#39;une solutisur la variable ou l&#39;heure, respectivement. Une caractéristique clé de l&#39;empreinte est que l&#39;exposition limitée à la sonde (par exemple, &quot;la cible unique cinétiques») des résultats de l&#39;échantillonnage uniforme de chaque nucléotide du polymère. 5 Dans cet article, vidéo, nous utilisons le domaine P4-P6 du ribozyme de Tetrahymena pour illustrer la préparation des échantillons d&#39;ARN et la détermination d&#39;un Mg (II)-médiée isothermes pliantes. Nous décrivons l&#39;utilisation du protocole bien connu l&#39;empreinte de radicaux hydroxyles qui nécessite H 2 O 2 (nous appelons cela la &quot;peroxydante« protocole) et une valeur, mais pas très connu, alternative qui utilise naturellement dissous O 2 (nous appelons cela «l&#39; oxydatif &quot;protocole). Un aperçu de la réduction des données, la transformation et les procédures de l&#39;analyse est présentée.

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

Ce protocole décrit comment quantifier les Mg (II)-dépendante de la formation de l&#39;ARN structure tertiaire par deux méthodes d&#39;empreinte radical hydroxyle.

Les changements d&#39;équilibre dans la structure de l&#39;ARN de surveillance par «peroxydation» et «oxydatif» Hydroxyl Footprinting Radical

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