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

Este trabalho foi suportado por concessões de NIH AG030709 / NIA e 07-65798 do Departamento de Saúde Pública da Califórnia. Reconhecemos Margaret M. Condron para peptídeo síntese e análise de aminoácidos, Dr. Elizabeth Neufeld F. para ajudar e apoiar os passos iniciais do projeto, Dr. Chi-Hong B. Chen para dar suporte e reagentes, e André, Dr. D . Ellington para discussões úteis.

Parte 1: preparação de proteína e cross-linking Inicialmente, a proteína usada para SELEX é pré-tratados com 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) para obter homogênea, agregar-free preparações, como descrito anteriormente 23. Esta etapa é necessária porque pré-formados agregados induzir a agregação rápida de proteínas amiloidogênicas, resultando em reprodutibilidade experimental pobres 24, e são indesejáveis ​​para a seleção de aptâmeros para...

<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>

O ponto de partida do processo SELEX é a síntese de uma biblioteca de oligonucleotídeos aleatórios tipicamente contendo 10 12 -10 15 seqüências. No DNA SELEX, esta biblioteca é usada diretamente depois de um pool de ssDNA é gerada, enquanto que no RNA SELEX, demonstrado aqui, a biblioteca ssDNA é convertido primeiro a um pool de RNA enzimaticamente por in-vitro de transcrição. Então, é realizada iterativamente SELEX que cada ciclo compõe a exposição e ligação de oligonucl...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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

Doença de Alzheimer (DA) é uma progressiva, dependente da idade desordem, neurodegenerativas com um curso insidioso que torna seu diagnóstico pré-sintomático difícil 1. AD diagnóstico definitivo é obtido post-mortem só, estabelecendo assim o diagnóstico, pré-sintomático precoce da AD é crucial para o desenvolvimento e administração de terapias eficazes 2,3. Β-amilóide-proteína (Aâ) é central para a patogênese AD. Solúvel, oligomérica assembléias Aâ Acredita-se que afetam a disfunção subjacente neurotoxicidade sináptica e perda de neurônios na AD 4,5. Várias formas de assembléias Aâ solúveis têm sido descritos, no entanto, suas inter-relações e relevância para a etiologia e patogenia AD são complexas e não bem compreendido 6. Ferramentas específicas de reconhecimento molecular pode desvendar as relações entre os conjuntos Aâ e facilitar a detecção e caracterização destes conjuntos no início do curso da doença antes que os sintomas surjam. Reconhecimento molecular comumente se baseia em anticorpos. No entanto, uma classe alternativa de ferramentas de reconhecimento molecular, aptâmeros, oferece vantagens importantes em relação aos anticorpos 7,8. Aptâmeros são oligonucleotides gerados por in-vitro de seleção: a evolução sistemática de ligantes por enriquecimento exponencial (SELEX) 9,10. SELEX é um processo iterativo que, semelhante à evolução darwinista, permite a amplificação de seleção, o enriquecimento, ea perpetuação de uma propriedade, por exemplo, ávido, específico, ligante de ligação (aptâmeros) ou atividade catalítica (ribozimas e DNAzymes). Apesar da emergência de aptâmeros como ferramentas em biotecnologia moderna e da medicina 11, eles têm sido subutilizados no campo amilóide. RNA poucos ou aptâmeros ssDNA foram selecionados contra diversas formas da proteína príon (PrP) 12-16. Um aptamer RNA recombinante contra gerada PrP bovina foi mostrado para reconhecer bovina PrP-β 17, um solúvel, oligomérica, β-folhas ricas variante conformacional de full-length PrP que forma fibrilas amilóides 18. Aptâmeros gerados usando formas monomérica e vários de β fibrilar 2-microglobulina (β 2 m) foram encontradas para ligar fibrilas de algumas outras proteínas amiloidogênicas além β 2 m fibrilas 19. Ylera et al. descrito aptâmeros RNA selecionada imobilizada contra monomérica Aβ40 20. Inesperadamente, esses aptâmeros ligado fibrilar Aβ40. Em conjunto, esses dados levantam várias questões importantes. Por que aptâmeros selecionados contra proteínas monoméricas reconhecer suas formas poliméricas? Poderia aptâmeros contra as formas monoméricas e / ou de proteínas oligoméricas amiloidogênicas ser obtido? Para responder a essas questões, buscou-se selecionar aptâmeros para covalentemente estabilizado oligomérica Aβ40 21 gerados usando foto-induzida cross-linking de proteínas não modificadas (picup) 22,23. Similar aos resultados anteriores 17,19,20, esses aptâmeros reagiu com fibrilas de Aâ e várias outras proteínas amiloidogênicas provável o reconhecimento de um amilóide potencialmente estrutural comum aptatope 21. Aqui, apresentamos a metodologia SELEX utilizados na produção destes aptâmeros 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

Aptâmeros são ribo-/deoxyribo-oligonucleotides curtas selecionados por<em&gt; In-vitro</em&gt; Métodos de evolução com base na afinidade por um alvo específico. Aptâmeros são ferramentas de reconhecimento molecular com o versátil aplicações terapêuticas, de diagnóstico e pesquisa. Demonstramos métodos de selecção dos aptâmeros para a proteína β-amilóide, o agente causador da doença de Alzheimer.

Seleção de Aptamers para proteína β-amilóide, o agente causador da doença de 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

Assaying Toxicidade β-amilóide utilizando um transgênico C. elegans Modelo

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

Neurociência

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

Preparação de fatias do hipocampo aguda de ratos e camundongos transgênicos para o Estudo das Alterações Synaptic durante o envelhecimento e amilóide Patologia

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

Detecção de placas neuríticas no rato da doença de Alzheimer Modelo

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

Ensaios olfativas para o Rato modelos de doenças neurodegenerativas

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

Infusão estereotáxica de oligomica beta-amilóide no ratinho Hippocampus

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

Por Quantificação Matérias: Caracterização de fenótipos na<em&gt; Drosophila</em&gt; Larval Junção Neuromuscular

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

Toxicidade do Amyloid-β42 de modelagem e neurodegeneração no cérebro adulto de Zebrafish

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

Na Vivo Único-molécula de rastreamento no Terminal do nervo Motor pré-sináptica da drosófila

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

Visualização dos depósitos β amiloide no cérebro humano com Matrix-assisted Laser dessorção/ionização de imagem espectrometria de massa

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

Rotulagem e imagem de placas amilóides no tecido cerebral usando a curcumina polifenóis naturais

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