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

Questo lavoro è stato sostenuto dalle concessioni dal National Institute of Health RO1-GM085130 e la National Science Foundation MCB0929394. Ringraziamo il Dott. Marion Schmidt per la sua ospitalità e per averci permesso di filmare nel suo laboratorio.

1. Preparazione dei reagenti Footprinting <ol><li> Preparare un tampone di reazione 10x contenente 100 cacodilato sodio mM, 1 mM EDTA, e 1 M KCl. Regolare il pH a 7,4. Filtrare il tampone usando un dispositivo di acetato 0,2 mM filtro (Nalgene). Nota: non RNA pipettare direttamente nel buffer 10x. </li><li> Preparare la miscela di reazione di titolazione per ogni reazione, come indicato nella tabella 1. Il volume del mix di titolazione (1x buffer e Mg (II) alla concentrazione desiderata) dovrebbe essere di 90 microlit...

<ol>
	<li>Tullius, T. D., Dombroski, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl+radical+"footprinting":+high-resolution+information+about+DNA-protein+contacts+and+application+to+lambda+repressor+and+Cro+protein.">Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 5469-5473 (1986).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron(II)-ethylenediaminetetraacetic+acid+catalyzed+cleavage+of+RNA+and+DNA+oligonucleotides:+similar+reactivity+toward+single-+and+double-stranded+forms.">Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms.</a> Biochemistry. 29, 1355-1361 (1990).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+higher+order+folding+of+a+catalytic+RNA+molecule.">Visualizing the higher order folding of a catalytic RNA molecule.</a> Science. 251, 401-407 (1991).</li><li>Sclavi, B., Sullivan, M., Chance, M. R., Brenowitz, M., Woodson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+folding+at+millisecond+intervals+by+synchrotron+hydroxyl+radical+footprinting.">RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.</a> Science. 279, 1940-1943 (1998).</li><li>Tullius, T. D., Dombroski, B. A., Churchill, M., Kam, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl+radical+footprinting:+a+high-resolution+method+for+mapping+protein-DNA+contacts.">Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts.</a> Methods. Enzym. 155, 537-558 (1987).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligoribonucleotide+synthesis+using+T7+RNA+polymerase+and+synthetic+DNA+templates.">Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.</a> Nucleic. Acids. Res. 15, 8783-8798 (1987).</li><li>Schlatterer, J. C., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementing+global+measures+of+RNA+folding+with+local+reports+of+backbone+solvent+accessibility+by+time+resolved+hydroxyl+radical+footprinting.">Complementing global measures of RNA folding with local reports of backbone solvent accessibility by time resolved hydroxyl radical footprinting.</a> Methods. 49, 142-147 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2001).</li><li>Zaug, A. J., Grosshans, C. A., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+endoribonuclease+activity+of+the+Tetrahymena+ribozyme:+enhanced+cleavage+of+certain+oligonucleotide+substrates+that+form+mismatched+ribozyme-substrate+complexes.">Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.</a> Biochemistry. 27, 8924-8931 (1988).</li><li>Knapp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+approaches+to+probing+of+RNA+secondary+and+tertiary+structure.">Enzymatic approaches to probing of RNA secondary and tertiary structure.</a> Methods. Enzym. 180, 192-212 (1989).</li><li>Slatko, B. E., Albright, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+gel+electrophoresis+for+sequencing.">Denaturing gel electrophoresis for sequencing.</a> Curr. Protoc. Mol. Biol. Chapter 7, Unit 7.6-Unit 7.6 (2001).</li><li>Das, R., Laederach, A., Pearlman, S. M., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAFA:+semi-automated+footprinting+analysis+software+for+high-throughput+quantification+of+nucleic+acid+footprinting+experiments.">SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.</a> RNA. 11, 344-354 (2005).</li><li>Simmons, K., Martin, J. S., Shcherbakova, I., Laederach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+and+analysis+of+kinetic+*OH+radical+footprinting+data+using+SAFA.">Rapid quantification and analysis of kinetic *OH radical footprinting data using SAFA.</a> Methods. Enzym. 468, 47-66 (2009).</li><li>Uchida, T., He, Q., Ralston, C. Y., Brenowitz, M., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+of+monovalent+and+divalent+ion+binding+in+the+folding+of+the+P4-P6+domain+of+the+Tetrahymena+ribozyme.">Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme.</a> Biochemistry. 41, 5799-5806 (2002).</li><li>Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+group+I+ribozyme+domain:+principles+of+RNA+packing.">Crystal structure of a group I ribozyme domain: principles of RNA packing.</a> Science. 273, 1678-1685 (1996).</li><li>Petri, V., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nucleic+acids+footprinting:+thermodynamic+and+kinetic+approaches.">Quantitative nucleic acids footprinting: thermodynamic and kinetic approaches.</a> Curr. Opin. Biotechnol. 8, 36-44 (1997).</li><li>Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+RNA+compaction:+insights+from+the+equilibrium+folding+pathway+of+the+P4-P6+RNA+domain+in+monovalent+cations.">Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations.</a> J. Mol. Biol. 343, 1195-1206 (2004).</li><li>Brenowitz, M., Senear, D. F., Shea, M. A., Ackers, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+DNase+footprint+titration:+a+method+for+studying+protein-DNA+interactions.">Quantitative DNase footprint titration: a method for studying protein-DNA interactions.</a> Methods. Enzym. 130, 132-181 (1986).</li><li>Shcherbakova, I., Mitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl-radical+footprinting+to+probe+equilibrium+changes+in+RNA+tertiary+structure.">Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure.</a> Methods. Enzym. 468, 31-46 (2009).</li><li>Howlett, G. J., Minton, A. P., Rivas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation+for+the+study+of+protein+association+and+assembly.">Analytical ultracentrifugation for the study of protein association and assembly.</a> Curr. Opinion. Chem. Biol. 10, 430-436 (2006).</li><li>McGinnis, J. L., Duncan, C. D., 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=High-throughput+SHAPE+and+hydroxyl+radical+analysis+of+RNA+structure+and+ribonucleoprotein+assembly.">High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.</a> Methods. Enzym. 468, 67-89 (2009).</li><li>Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+large+RNA+molecules+with+knowledge-based+potentials+and+structural+filters.">Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters.</a> RNA. 15, 189-199 (2009).</li></ol>

Footprinting radicale ossidrile è uno strumento prezioso per valutare l&#39;area solvente superficie accessibile di acidi nucleici. Formazione qualitativa e quantitativa della struttura terziaria 14 può essere seguita in funzione di parametri quali il tipo e la concentrazione di ioni, pH, temperatura, proteine ​​di legame o pieghevoli co-fattori. La combinazione vincente di un protocollo di dritto in avanti ed economico e l&#39;accessibilità conseguente solvente e le informazioni su un pieghevole a sin...

<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

Molecole di RNA svolgono un ruolo essenziale in biologia. Oltre alla trasmissione di informazioni genetiche, l&#39;RNA può piegare in unico strutture terziarie che compie un ruolo biologico specifico come regolatore, legante o catalizzatore. Informazioni sulla formazione terziaria contatto è essenziale per capire la funzione di molecole di RNA. I radicali idrossile (OH •) sono le sonde della struttura degli acidi nucleici a causa della loro elevata reattività e piccole dimensioni. 1 Quando utilizzato come sonda footprinting, i radicali idrossili mappare la superficie solvente accessibile della dorsale fosfodiestereo di DNA 1 e 2 con RNA sottile come singolo nucleotide risoluzione. Idrossile footprinting radicale può essere utilizzato per identificare i nucleotidi all&#39;interno di una superficie di contatto intermolecolari, ad esempio nel DNA-proteina 1 e RNA-proteina complessi. Equilibrio 3 e 4 transizioni cinetica può essere determinato da condurre footprinting radicale ossidrile in funzione di una soluzione moltoil variabile o il tempo, rispettivamente. Una caratteristica fondamentale di footprinting è che l&#39;esposizione limitata alla sonda (per esempio, &#39;single-hit cinetica&#39;) i risultati nel campionamento uniforme di ogni nucleotide del polimero 5. In questo articolo il video, si usa il P4-P6 dominio del ribozima Tetrahymena per illustrare la preparazione dei campioni di RNA e la determinazione di un (II)-mediata isoterme Mg pieghevole. Descriviamo l&#39;uso del noto protocollo di idrossile footprinting radicale che richiede H 2 O 2 (chiamiamo questo il protocollo &#39;perossidativi&#39;) e un valore, ma poco noto, alternativa che utilizza naturalmente disciolto O 2 (lo chiamiamo la &#39; ossidativo &#39;protocollo). Una panoramica della riduzione dei dati, la trasformazione e procedure di analisi è presentato.

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

Questo protocollo descrive come quantificare il Mg (II)-dipendente formazione della struttura terziaria dell&#39;RNA con due metodi di footprinting radicali idrossili.

Cambiamenti di equilibrio nella struttura di monitoraggio RNA da &#39;perossidativi&#39; e &#39;ossidativo&#39; Footprinting radicale ossidrile

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

Research

JoVE Journal

Biology

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Metodi di singola molecola per monitorare l&#39;evoluzione a doppio strato: Proprietà meccaniche

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Ricerca

Biologia

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Monitoraggio Cambiamenti dei livelli di calcio mitocondriale durante l&#39;apoptosi mediante un sensore di calcio geneticamente codificato

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

A lungo termine del test di tossicità letale con il crostaceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Chromatin Isolation by RNA Purification ChIRP

Isolamento di cromatina Purificazione RNA (disturbo)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

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 ...

Analysis of Oxidative Stress in Zebrafish Embryos

Analisi dello Stress Ossidativo in embrioni di zebrafish

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

Ricostituzione funzionale e attività Canale Misure di purificata di tipo selvatico e mutante CFTR proteine

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

Rilevamento delle modifiche droga indotte recettore post-internalizzazione Tratta di Analisi Colocalizational

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Fecale Analisi glucocorticoidi: non invasivo di monitoraggio surrenale in equidi

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...