Name: selection of aptamers for amyloid &beta;-protein, the causative agent of alzheimer&#39;s disease
Uploaded: 2010-05-13T17:00:00
Description: Watch this Scientific Journal Video about Selection of Aptamers for Amyloid ÃŽÂ²-Protein, the Causative Agent of Alzheimer's Disease at JoVE.com

Ce travail a été soutenu par des subventions du NIH AG030709 / NIA et 07-65798 du California Department of Public Health. Nous reconnaissons Margaret M. Condron pour la synthèse peptidique et de l&#39;analyse des acides aminés, le Dr Elizabeth F. Neufeld pour les aider et de soutenir les premières étapes du projet, le Dr Chi-Hong Chen B. de fournir un soutien et des réactifs, et le Dr Andrew D . Ellington pour les discussions utiles.

Partie 1: préparation de protéines et de réticulation Initialement, la protéine utilisée pour SELEX est prétraitée avec 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) pour obtenir homogène, sans agrégat préparations, comme décrit précédemment 23. Cette étape est nécessaire car pré-formé des agrégats induire l&#39;agrégation rapide des protéines amyloïdogénique, résultant en mauvaise reproductibilité expérimentale 24 ans, et sont indésirables pour la s?...

<ol>
	<li>Monien, B. H., Apostolova, L. G., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+diagnostics+and+therapeutics+for+Alzheimer's+disease-how+early+can+we+get+there.">Early diagnostics and therapeutics for Alzheimer's disease-how early can we get there.</a> Expert. Rev. Neurother. 6, 1293-1306 (2006).</li><li>Nestor, P. J., Scheltens, P., Hodges, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+early+detection+of+Alzheimer's+disease.">Advances in the early detection of Alzheimer's disease.</a> Nat. Med. 10, S34-S41 (2004).</li><li>Kawas, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Early+Alzheimer's+disease.">Clinical practice. Early Alzheimer's disease.</a> N. Engl. J. Med. 349, 1056-1063 (2003).</li><li>Haass, C., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+protein+oligomers+in+neurodegeneration:+lessons+from+the+Alzheimer's+amyloid+&#946;-peptide.">Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid &#946;-peptide.</a> Nat. Rev. Mol. Cell. Biol. 8, 101-112 (2007).</li><li>Kirkitadze, M. D., Bitan, G., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paradigm+shifts+in+Alzheimer's+disease+and+other+neurodegenerative+disorders:+the+emerging+role+of+oligomeric+assemblies.">Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies.</a> J. Neurosci. Res. 69, 567-577 (2002).</li><li>Rahimi, F., Shanmugam, A., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+pre-fibrillar+protein+assemblies+in+Alzheimer's+disease+and+related+disorders.">Structure-function relationships of pre-fibrillar protein assemblies in Alzheimer's disease and related disorders.</a> Curr. Alzheimer Res. 5, 319-341 (2008).</li><li>Jayasena, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers:+an+emerging+class+of+molecules+that+rival+antibodies+in+diagnostics.">Aptamers: an emerging class of molecules that rival antibodies in diagnostics.</a> Clin. Chem. 45, 1628-1650 (1999).</li><li>Bunka, D. H., Stockley, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamers+come+of+age+-+at+last.">Aptamers come of age - at last.</a> Nat. Rev. Microbiol. 4, 588-596 (2006).</li><li>Ellington, A. D., Szostak, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+selection+of+RNA+molecules+that+bind+specific+ligands.">In vitro selection of RNA molecules that bind specific ligands.</a> Nature. 346, 818-822 (1990).</li><li>Tuerk, C., Gold, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+evolution+of+ligands+by+exponential+enrichment:+RNA+ligands+to+bacteriophage+T4+DNA+polymerase.">Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.</a> Science. 249, 505-510 (1990).</li><li>Lee, J. F., Stovall, G. M., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aptamer+therapeutics+advance.">Aptamer therapeutics advance.</a> Curr. Opin. Chem. Biol. 10, 282-289 (2006).</li><li>Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+specifically+interact+with+the+prion+protein+PrP.">RNA aptamers specifically interact with the prion protein PrP.</a> J. Virol. 71, 8790-8797 (1997).</li><li>Bibby, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+novel+in+vitro+selection+technique+to+isolate+and+characterise+high+affinity+DNA+aptamers+binding+mammalian+prion+proteins.">Application of a novel in vitro selection technique to isolate and characterise high affinity DNA aptamers binding mammalian prion proteins.</a> J. Virol. Methods. 151, 107-115 (2008).</li><li>Rhie, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+2'-fluoro-RNA+aptamers+that+bind+preferentially+to+disease-associated+conformations+of+prion+protein+and+inhibit+conversion.">Characterization of 2'-fluoro-RNA aptamers that bind preferentially to disease-associated conformations of prion protein and inhibit conversion.</a> J. Biol. Chem. 278, 39697-39705 (2003).</li><li>King, D. J., Safar, J. G., Legname, G., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thioaptamer+interactions+with+prion+proteins:+sequence-specific+and+non-specific+binding+sites.">Thioaptamer interactions with prion proteins: sequence-specific and non-specific binding sites.</a> J. Mol. Biol. 369, 1001-1014 (2007).</li><li>Proske, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion-protein-specific+aptamer+reduces+PrPSc+formation.">Prion-protein-specific aptamer reduces PrPSc formation.</a> ChemBioChem. 3, 717-725 (2002).</li><li>Murakami, K., Nishikawa, F., Noda, K., Yokoyama, T., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-bovine+prion+protein+RNA+aptamer+containing+tandem+GGA+repeat+interacts+both+with+recombinant+bovine+prion+protein+and+its+&#946;+isoform+with+high+affinity.">Anti-bovine prion protein RNA aptamer containing tandem GGA repeat interacts both with recombinant bovine prion protein and its &#946; isoform with high affinity.</a> Prion. 2, 73-80 (2008).</li><li>Luhrs, T., Zahn, R., Wuthrich, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+formation+by+recombinant+full-length+prion+proteins+in+phospholipid+bicelle+solutions.">Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions.</a> J. Mol. Biol. 357, 833-841 (2006).</li><li>Bunka, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+and+characterization+of+RNA+aptamers+specific+for+amyloid+fibril+epitopes.">Production and characterization of RNA aptamers specific for amyloid fibril epitopes.</a> J. Biol. Chem. 282, 34500-34509 (2007).</li><li>Ylera, F., Lurz, R., Erdmann, V. A., Furste, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+RNA+aptamers+to+the+Alzheimer's+disease+amyloid+peptide.">Selection of RNA aptamers to the Alzheimer's disease amyloid peptide.</a> Biochem. Biophys. Res. Commun. 290, 1583-1588 (2002).</li><li>Rahimi, F., Murakami, K., Summers, J. L., Chen, C. H., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+generated+against+oligomeric+A&#946;40+recognize+common+amyloid+aptatopes+with+low+specificity+but+high+sensitivity.">RNA aptamers generated against oligomeric A&#946;40 recognize common amyloid aptatopes with low specificity but high sensitivity.</a> PLoS ONE. 4, e7694-e7694 (2009).</li><li>Bitan, G., Lomakin, A., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+&#946;-protein+oligomerization:+prenucleation+interactions+revealed+by+photo-induced+cross-linking+of+unmodified+proteins.">Amyloid &#946;-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.</a> J. Biol. Chem. 276, 35176-35184 (2001).</li><li>Rahimi, F., Maiti, P., Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+cross-linking+of+unmodified+proteins+(PICUP)+applied+to+amyloidogenic+peptides.">Photo-induced cross-linking of unmodified proteins (PICUP) applied to amyloidogenic peptides.</a> J. Vis. Exp. , (2009).</li><li>Bitan, G., Fradinger, E. A., Spring, S. M., Teplow, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+protein+oligomers-what+you+see+is+not+always+what+you+get.">Neurotoxic protein oligomers-what you see is not always what you get.</a> Amyloid. 12, 88-95 (2005).</li><li>Bitan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+study+of+metastable+amyloidogenic+protein+oligomers+by+photo-induced+cross-linking+of+unmodified+proteins.">Structural study of metastable amyloidogenic protein oligomers by photo-induced cross-linking of unmodified proteins.</a> Methods Enzymol. 413, 217-236 (2006).</li><li>Chen, C. H., Chernis, G. A., Hoang, V. Q., Landgraf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+heregulin+signaling+by+an+aptamer+that+preferentially+binds+to+the+oligomeric+form+of+human+epidermal+growth+factor+receptor-3.">Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3.</a> Proc. Natl. Acad. Sci. USA. 100, 9226-9231 (2003).</li><li>Adams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lab math: a handbook of measurements, calculations, and other quantitative skills for use at the bench. , (2003).</li><li>Gopinath, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+developed+for+SELEX.">Methods developed for SELEX.</a> Anal. Bioanal. Chem. 387, 171-182 (2007).</li><li>Takahashi, T., Tada, K., Mihara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+aptamers+selected+against+amyloid+&#946;-peptide+(A&#946;)+inhibit+the+aggregation+of+A&#946;.">RNA aptamers selected against amyloid &#946;-peptide (A&#946;) inhibit the aggregation of A&#946;.</a> Mol. Biosyst. 5, 986-991 (2009).</li></ol>

Le point de départ du processus SELEX est la synthèse d&#39;une bibliothèque d&#39;oligonucléotides aléatoires contenant typiquement 10 12 -10 15 séquences. Dans l&#39;ADN SELEX, cette bibliothèque est utilisée directement après une piscine ADNss est généré, alors que dans l&#39;ARN SELEX, démontré ici, la bibliothèque ADNss est convertie d&#39;abord à un pool d&#39;ARN enzymatiquement par transcription in vitro. Puis, SELEX est réalisé itérativement lequel chaq...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>A&beta;40</td>
<td>&nbsp;</td>
<td>UCLA Biopolymers Laboratory</td>
<td>&nbsp;</td>
<td>Lyophilized powder</td>
</tr>
<tr>
<td>MX5 Automated-S Microbalance</td>
<td>&nbsp;</td>
<td>Mettler Toledo</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silicon-coated, 1.6-ml tubes</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19033 or C19035</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)</td>
<td>&nbsp;</td>
<td>TCI America</td>
<td>H0424</td>
<td>Use in a fume hood.</td>
</tr>
<tr>
<td>Ammonium persulfate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A-7460</td>
<td>Vortex until the solution is clear. APS is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Tris(2,2-bipridyl)dichlororuthenium(II) hexahydrate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>224758-1G</td>
<td>Vortex until the solution is clear. Cover the RuBpy tube with foil to protect the reagent from ambient light. RuBpy is prepared freshly each time and should be used within 48 h.</td>
</tr>
<tr>
<td>Dithiothreitol (DTT)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>43815</td>
<td>&nbsp;</td>
<tr>
<td>D-Salt&trade; Excellulose&trade; desalting columns</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>20449</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium acetate	</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A637-500</td>
<td>&nbsp;</td>
<tr>
<td>Silicon-coated, 0.6-ml tubes</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19063</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tricine Gels (10&#x2013;20%)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6625B0X</td>
<td>10-well; mini size (8 cm X 8 cm); 25 &mu;l loading volume per well; separation range 5 kDa to 40 kDa</td>
</tr>
<tr>
<td>Quartz cuvette</td>
<td>&nbsp;</td>
<td>Hellma</td>
<td>105.250-QS</td>
<td>&nbsp;</td>
<tr>
<td>Beckman DU 640 spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ssDNA library</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>The library was designed to contain 49 random nucleotides flanked by two constant regions containing primer-binding and cloning sites: 
5'-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C (N:25:25:25:25%) (N)49 G TCC GTT CGG GAT CCT C-3'</td>
</tr>
<tr>
<td>Taq DNA polymerase</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71160</td>
<td>Recombinant Thermus aquaticus DNA Polymerase supplied with 10&#215; PCR Buffer and a separate tube of 25 mM MgCl2 for routine PCR.</td>
</tr>
<tr>
<td>PCR Nucleotide Mix, 10 mM solution</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>77212</td>
<td>(10 mM each dATP, dCTP, dGTP, dTTP)</td>
</tr>
<tr>
<td>Forward primer</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>5'-TAA TAC GAC TCA CTA TAG GGA ATT CCG CGT GTG C-3'</td>
</tr>
<tr>
<td>Reverse primer</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies</td>
<td>Custom-ordered</td>
<td>5'-GAG GAT CCC GAA CGG AC-3'</td>
</tr>
<tr>
<td>Thermal cycler</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>Techne TC-312</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick PCR Purification Kit (50)</td>
<td>&nbsp;</td>
<td>QIAGEN</td>
<td>28104</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>CA3510-8</td>
<td>&nbsp;</td>
<tr>
<td>Conical, sterile 1.6-ml tubes with caps attached with O-rings</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C19040-S</td>
<td>&nbsp;</td>
<tr>
<td>RiboMAX&trade; Large Scale RNA Production System&#x2013;T7</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>P1300</td>
<td>The kit contains: 120 &mu;l Enzyme Mix (RNA polymerase, recombinant RNasin&reg; ribonuclease inhibitor and recombinant inorganic pyrophosphatase); 240 &mu;l transcription 5 buffer; 100 &mu;l each of 4 rNTPs, 100 mM; 110 U RQ1 RNase-free DNase, 1 U/&mu;l; 10 &mu;l linear control DNA, 1 mg/ml; 1 ml 3M sodium acetate (pH 5.2); 1.25 ml nuclease-Free water</td>
</tr>
<tr>
<td>&alpha;-32P-cytidine 5'-triphosphate, 250 &mu;Ci (9.25 MBq), </td>
<td>&nbsp;</td>
<td>Perkin Elmer</td>
<td>BLU008H250UC</td>
<td>Specific Activity: 3000 Ci (111 TBq)/mmol, 50 mM Tricine (pH 7.6)</td>
</tr>
<tr>
<td>Citrate-saturated phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.7)</td>
<td>&nbsp;</td>
<td>Sigma (Fluka)</td>
<td>77619</td>
<td>&nbsp;</td>
<tr>
<td>Chloroform:Isoamyl alcohol (24:1)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C0549</td>
<td>&nbsp;</td>
<tr>
<td>Absolute ethanol for molecular biology</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E7023</td>
<td>&nbsp;</td>
<tr>
<td>Z216-MK refrigerated microcentrifuge</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>C0216-MK</td>
<td>&nbsp;</td>
<tr>
<td>illustra ProbeQuant&trade; G-50 Micro Columns</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>Obtained from Fisher Scientific (45-001-487)</td>
<td>Prepacked with Sephadex&trade; G-50 DNA Grade and pre-equilibrated in STE buffer containing 0.15% Kathon as Biocide</td>
</tr>
<tr>
<td>Triathler Bench-top Scintillation counter</td>
<td>&nbsp;</td>
<td>Hidex Oy, Turku, Finland</td>
<td>Triathler LSC Model: 425-034</td>
<td>&nbsp;</td>
<tr>
<td>Novex&reg; TBE-Urea Sample Buffer (2&#215;)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6876</td>
<td>&nbsp;</td>
<tr>
<td>6% TBE-Urea Gels 1.0 mm, 10 wells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC6865BOX</td>
<td>&nbsp;</td>
<tr>
<td>Novex&reg; TBE Running Buffer (5&#215;)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6675</td>
<td>&nbsp;</td>
<tr>
<td>Radioactivity decontaminant</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>04-355-67</td>
<td>&nbsp;</td>
<tr>
<td>Gel-loading tips</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>P3080</td>
<td>&nbsp;</td>
<tr>
<td>XCell SureLock Mini-Cell</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>XCell SureLock Mini-Cell</td>
</tr>
<tr>
<td>Autoradiography film</td>
<td>&nbsp;</td>
<td>Denville Scientific</td>
<td>E3018</td>
<td>Use in complete darkness</td>
</tr>
<tr>
<td>Autoradiography film, Hyperfilm&trade; ECL</td>
<td>&nbsp;</td>
<td>Amersham Biosciences</td>
<td>RPN3114K</td>
<td>Can be used under red safe light.</td>
</tr>
<tr>
<td>Membrane discs</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>GSWP02500</td>
<td>Mixed cellulose ester, hydrophilic, 0.22-&mu;m disc membranes</td>
</tr>
<tr>
<td>Fritted glass support base for 125-ml flask</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>26316-696</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>08-757-11YZ</td>
<td>&nbsp;</td>
<tr>
<td>Urea	</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>AC32738-0050</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>118430010</td>
<td>&nbsp;</td>
<tr>
<td>Glycogen</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G1767</td>
<td>&nbsp;</td>
<tr>
<td>2-Propanol for molecular biology</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I9516</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant RNase inhibitor</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71571</td>
<td>&nbsp;</td>
<tr>
<td>ImProm-II&trade;Reverse Transcription System</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A3802</td>
<td>&nbsp;</td>
<tr>
<td>Recombinant RNase inhibitor</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>71571</td>
<td>&nbsp;</td>
<tr>
<td>RapidRun&trade; Loading Dye</td>
<td>&nbsp;</td>
<td>USB Corporation</td>
<td>77524</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

selection of aptamers for amyloid &beta;-protein, the causative agent of alzheimer&#39;s disease

La maladie d&#39;Alzheimer (MA) est une progressive, dépendant de l&#39;âge, maladie neurodégénérative avec une évolution insidieuse qui rend son diagnostic présymptomatique difficiles 1. AD diagnostic définitif est réalisé l&#39;autopsie seulement, établissant ainsi présymptomatique, le diagnostic précoce de la MA est crucial pour développer et administrer des traitements efficaces 2,3. Amyloïde β-protéine (Aß) est au centre de la pathogenèse AD. Soluble, oligomérique assemblées Aßi auraient des effets sur la neurotoxicité dysfonction sous-jacente synaptique et la perte de neurones dans l&#39;AD 4,5. Diverses formes de Aß solubles assemblées ont été décrits, cependant, leurs interrelations et de la pertinence de l&#39;étiologie et la pathogenèse AD sont complexes et mal comprises 6. Des outils spécifiques de reconnaissance moléculaire peut démêler les relations entre les assemblées Aßi et de faciliter la détection et la caractérisation de ces assemblées au début de l&#39;évolution de la maladie avant que les symptômes apparaissent. La reconnaissance moléculaire s&#39;appuie souvent sur des anticorps. Cependant, une autre classe d&#39;outils de reconnaissance moléculaire, des aptamères, offre des avantages importants par rapport aux anticorps 7,8. Les aptamères sont des oligonucléotides générés par la sélection in vitro: l&#39;évolution systématique des ligands par enrichissement exponentiel (SELEX) 9,10. SELEX est un processus itératif qui, semblable à l&#39;évolution darwinienne, permet la sélection, d&#39;amplification, d&#39;enrichissement, et la perpétuation d&#39;une propriété, par exemple, avide, ligand spécifique, contraignant (aptamères) ou de l&#39;activité catalytique (ribozymes et ADNzymes). Malgré l&#39;émergence d&#39;aptamères comme outils de la biotechnologie moderne et la médecine 11, ils ont été sous-utilisés dans le domaine de l&#39;amyloïde. Peu ou ARN aptamères ADNss ont été sélectionnés contre les différentes formes de protéines du prion (PrP) 12-16. Un aptamère ARN générés contre la PrP recombinante bovine a été montré à reconnaître la PrP bovine β-17, une forme soluble, oligomérique, β-feuille riche en variantes de conformation de la PrP pleine longueur qui forme des fibrilles amyloïdes 18. Les aptamères générés en utilisant les formes monomériques et plusieurs de β 2-microglobuline fibrillaire (β 2 m) ont été trouvés pour lier les fibrilles de certaines autres protéines amyloïdogéniques dehors β 2 m 19 fibrilles. Ylera et al. décrit aptamères d&#39;ARN sélectionnés contre immobilisé monomère Aß40 20. De façon inattendue, ces aptamères liés fibrillaire Aß40. Au total, ces données soulèvent plusieurs questions importantes. Pourquoi aptamères sélectionnés contre des protéines monomériques reconnaître leurs formes polymères? Pourriez aptamères contre les formes monomères et / ou oligomères de protéines amyloïdogéniques être obtenus? Pour répondre à ces questions, nous avons tenté de sélectionner des aptamères pour covalente stabilisée oligomères Aß40 21 généré en utilisant la photo-induite de réticulation des protéines non modifiées (PICUP) 22,23. Similaires aux constatations antérieures 17,19,20, ces aptamères ont réagi avec des fibrilles de Aß et de plusieurs autres protéines amyloïdogéniques susceptible de reconnaître une substance amyloïde potentiellement communes structurelles aptatope 21. Ici, nous présentons la méthodologie utilisée dans la production SELEX de ces aptamères 21.

Watch this Scientific Journal Video about Selection of Aptamers for Amyloid ÃŽÂ²-Protein, the Causative Agent of Alzheimer's Disease at JoVE.com

Selection of Aptamers for Amyloid ÃƒÅ½Ã‚Â²-Protein, the Causative Agent of Alzheimer's Disease

Les aptamères sont sélectionnés par ribo-/deoxyribo-oligonucleotides courte<em&gt; In-vitro</em&gt; Les méthodes basées sur l&#39;évolution affinité pour une cible spécifique. Les aptamères sont des outils de reconnaissance moléculaire avec polyvalente applications thérapeutiques, diagnostiques et de recherche. Nous démontrons méthodes de sélection des aptamères pour amyloïde β-protéine, l&#39;agent causal de la maladie d&#39;Alzheimer.

Sélection d&#39;aptamères pour amyloïde β-protéine, l&#39;agent causal de la maladie d&#39;Alzheimer

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Research

JoVE Journal

Neuroscience

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Assaying ÃƒÅ½Ã‚Â²-amyloid Toxicity using a Transgenic C. elegans Model

Dosage β-amyloïde toxicité utilisant un transgéniques C. elegans</emModèle>

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Recherche

Neurosciences

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Préparation des coupes d&#39;hippocampe aiguë chez des rats et des souris transgéniques pour l&#39;étude des altérations synaptiques au cours du vieillissement et de l&#39;amylose pathologie

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

La détection de plaques séniles dans le modèle d&#39;Alzheimer La maladie de la souris

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

Olfactory Assays for Mouse Models of Neurodegenerative Disease

Les tests olfactifs pour les modèles murins de maladies neurodégénératives

In many neurodegenerative diseases and particularly in Parkinson&#8217;s disease, deficits in olfaction are reported to occur early in the disease process ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Infusion stéréotaxique des oligomères bêta-amyloïde dans les Hippocampus souris

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Pourquoi Quantification Matters: Caractérisation des phénotypes au<em&gt; Drosophila</em&gt; Larvaire jonction neuromusculaire

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modeling Amyloid-ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Modélisation de toxicité amyloïde-β42 et neurodégénérescence dans le cerveau de poisson-zèbre adulte

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In Vivo Simple-molécule suivi du terminal de nerf moteur présynaptique Drosophila

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Visualization of Amyloid ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â² Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Visualisation des dépôts β-amyloïde dans le cerveau humain avec Laser assistée par matrice désorption-ionisation d’imagerie de la spectrométrie de masse

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Étiquetage et imagerie des plaques amyloïdes dans les tissus cérébraux à l'aide de la curcumine polyphénol naturelle

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...