Name: chemoselective modification of viral surfaces via bioorthogonal click chemistry
Uploaded: 2012-08-19T14:00:00
Description: Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

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Se tabell 1 for utarbeidelse av alle medier, buffere og løsninger refereres til i denne protokollen. 1. Produksjon av Aha-merket Adenovirus <ol><li> Forbered elleve 100-mm vevskulturstudier retter av humane embryonale nyre celler (HEK 293) opprettholdes i HEK 293 cellevekst medium (se tabell 1) ved 37 ° C til de har nådd 80 - 90% confluency. (Merk:. Kulturer med confluency over 90% kan være vanskelig å infisere og kan ikke produsere virus som ønsket)...

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	<li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Attachment+of+Small+Molecule+Effector+Functionality+to+Human+Adenoviruses+Facilitates+Gene+Delivery+to+Cancer+Cells.">Chemoselective Attachment of Small Molecule Effector Functionality to Human Adenoviruses Facilitates Gene Delivery to Cancer Cells.</a> J. Am. Chem. Soc. 132, 13615-13617 (2010).</li><li>Banerjee, P. S., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemoselective+Modification+of+Viral+Proteins+Bearing+Metabolically+Introduced+"Clickable"+Amino+Acids+and+Sugars.">Chemoselective Modification of Viral Proteins Bearing Metabolically Introduced "Clickable" Amino Acids and Sugars.</a> Methods Mol. Biol. 751, 55-66 (2011).</li><li>Waehler, R., Russell, S. J., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+targeted+viral+vectors+for+gene+therapy.">Engineering targeted viral vectors for gene therapy.</a> Nat. Rev. Genet. 8, 573-587 (2007).</li><li>Magnusson, M. K., Hong, S. S., Henning, P., Boulanger, P., Lindholm, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Retargeting+of+Adenovirus+Vectors:+Functionality+of+Targeting+Ligands+and+Their+Influence+on+Virus+Viability.">Genetic Retargeting of Adenovirus Vectors: Functionality of Targeting Ligands and Their Influence on Virus Viability.</a> J. Gene Med. 4, 356-370 (2002).</li><li>Henning, P., Lundgren, E., Carlsson, M., Frykholm, K., Johannisson, J., Magnusson, M. K., TÃ¥ng, E., Franqueville, L., Hong, S. S., Lindholm, L., Boulanger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+Type+5+Fiber+Knob+Domain+has+a+Critical+Role+in+Fiber+Protein+Synthesis+and+Encapsidation.">Adenovirus Type 5 Fiber Knob Domain has a Critical Role in Fiber Protein Synthesis and Encapsidation.</a> J. Gen. Virol. 87, 3151-3160 (2006).</li><li>Zhang, M. M., Tsou, L. K., Charron, G., Raghavan, A. S., Hang, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tandem+fluorescence+imaging+of+dynamic+S-acylation+and+protein+turnover.">Tandem fluorescence imaging of dynamic S-acylation and protein turnover.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 8627-8632 (2010).</li><li>Banerjee, P. S., Ostapchuk, P., Hearing, P., Carrico, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unnatural+amino+acid+incorporation+onto+adenoviral+(Ad)+coat+proteins+facilitates+chemoselective+modification+and+retargeting+of+Ad+type+5+vectors.">Unnatural amino acid incorporation onto adenoviral (Ad) coat proteins facilitates chemoselective modification and retargeting of Ad type 5 vectors.</a> J. Virol. 85, 7546-7554 (2011).</li><li>Hong, V., Presolski, S. I., Ma, C., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+Optimization+of+Copper-Catalyzed+Azide-Alkyne+Cycloaddition+for+Bioconjugation.">Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation.</a> Angew. Chem. Int. Ed. 48, 9879-9883 (2009).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+Chemistry+and+Bioorthogonal+Reactions:+Unprecedented+Selectivity+in+the+Labeling+of+Biological+Molecules.">Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules.</a> Biochemistry. 48, 6571-6584 (2009).</li><li>Agard, N. J., Prescher, J. A., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Strain-Promoted+[3+++2]+Azide-Alkyne+Cycloaddition+for+Covalent+Modification+of+Biomolecules+in+Living+Systems.">A Strain-Promoted [3 + 2] Azide-Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems.</a> J. Am. Chem. Soc. 46, 15046-15047 (2004).</li><li>Dommerholt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Readily+Accessible+Bicyclononynes+for+Bioorthogonal+Labeling+and+Three-Dimensional+Imaging+of+Living+Cells.">Readily Accessible Bicyclononynes for Bioorthogonal Labeling and Three-Dimensional Imaging of Living Cells.</a> Angew. Chem. Int. Ed. 49, 9422-9425 (2010).</li><li>Link, A. J., Vink, M. K. S., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+the+functionalizable+methionine+surrogate+azidohomoalanine+via+copper-catalyzed+diazotransfer.">Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazotransfer.</a> Nat. Protoc. 2, 1879-1883 (2007).</li></ol>

Utviklingen av chemoselective og bioorthogonal klikk reaksjoner, inkludert de av azider, er en hurtig utviklende område av forskning, og deretter er det et økende antall av disse reaksjonene å velge fra for bioconjugation applikasjoner. Vi har begrenset omfanget av denne protokollen til å omfatte bare to metoder som ble valgt på grunn av nytten sin i vår egen lab og kommersiell tilgjengelighet av alle reagenser. I den første slike rute - stamme-forfremmet azide-alkyne cycloaddition (S...

<table border="1"><tbody><tr><td> Navn på reagensen </td><td> Firma </td><td> Katalognummer </td></tr><tr><td> Adenovirus type 5 (AD5) inneholder en GFP transgene </td><td> BCBC </td><td> 391 </td></tr><tr><td> Humane embryonale nyre (HEK 293 celler) </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td> Dulbecco modifiserte Eagle Medium (DMEM), Høy Glukose </td><td> Invitrogen </td><td> 11965-092 </td></tr><tr><td> Dulbecco modifiserte Eagle Medium, nei metionin, ingen Cysteine ​​(DMEM-Met /-Cys), høye glukose </td><td> Invitrogen </td><td> 21013-024 </td></tr><tr><td> Bovine kalveserum </td><td> Invitrogen </td><td> 16170-078 </td></tr><tr><td> Penicillin - Streptomycin 100 × Solution (Pen Strep) </td><td> Invitrogen </td><td> 15140-122 </td></tr><tr><td> 0,5% Trypsin-EDTA (10 ×) </td><td&gt; Invitrogen </td><td> 15400-054 </td></tr><tr><td> 100 mm Cell Culture Dish, vev-kultur behandlet polystyren </td><td> BD Falcon </td><td> 353003 </td></tr><tr><td> Cellekultur CO 2 inkubator </td><td></td><td></td></tr><tr><td> Hemocytometer </td><td></td><td></td></tr><tr><td> Biosikkerhet kabinett </td><td></td><td></td></tr><tr><td> Steril sprøyte filter (0,2 mikrometer, celluloseacetat) </td><td> VWR </td><td> 28145-477 (NA), 514-0061 (Europa) </td></tr><tr><td> Avanti JE sentrifuge </td><td> Beckman Coulter </td><td> 369001 </td></tr><tr><td> Koniske rør (50 ml, sterilt) </td><td> BD Falcon </td><td> 352098 </td></tr><tr><td> Tube, Thinwall, Ultra-Clear, 13,2 ml, 14 x 89 mm </td><td> Beckman </td><td> 344059 </td></tr><tr><td> Ultrasentrifuger utstyrt med en SW 41 og SW 60 rotor </td><td> Beckman </td><td></td></tr><tr><td> Eppendorf Biopur Safe-Lock Rør, 1,5 ml </td><td> Eppendorf </td><td> 0030 121.589 </td></tr><tr><td> Sentrifuger 5418 </td><td> Eppendorf </td><td> 5418 </td></tr><tr><td> Centri-Sep gel filtrering spin kolonner </td><td> Princeton Separasjoner </td><td> CS-901 </td></tr><tr><td> Steril nål, 18 gauge </td><td></td><td></td></tr><tr><td> Nitrogen hanske bag (hvis deoxygenated CuAAC skal utføres) </td><td></td><td></td></tr><tr><td> Dewar kolbe </td><td></td><td></td></tr><tr><td> Flytende nitrogen </td><td></td><td></td></tr><tr><td> Elektroforese cellen </td><td></td><td></td></tr><tr><td> Fluoriserende gel skanner </td><td></td><td></td></tr><tr><td> Klar Gel Tris-HCl Gel </td><td> Bio-Rad </td><td> 161-1105 </td></tr><tr><td> L-Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td> Jena biovitenskap </td><td> CLK-AA005 </td><td></td></tr><tr><td> Peracetylated N-azidoacetylgalactosamine (Ac 4 GalNAz) </td><td> Invitrogen </td><td> C33365 </td></tr><tr><td> Thermo Scientific </td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich </td><td> A7480 </td><td></td></tr><tr><td> Bathophenanthroline disulfonic syre (BDA) disodium salt </td><td> MP Biomedicals </td><td> 0215011201 </td></tr><tr><td> Dimethyl sulfoxide (DMSO) </td><td></td><td></td></tr><tr><td> Metanol </td><td></td><td></td></tr><tr><td> Tris base (tris (hydroxymethyl) aminomethane) </td><td></td><td></td></tr><tr><td> Dinatriumfosfat (Na2 HPO 4) </td><td></td><td></td></tr><tr><td> Fosfatbufret saltvann (PBS) </td><td></td><td></td></tr><tr><td> 1M HCl </td><td></td><td></td></tr><tr><td> Glyserol </td><td></td><td></td></tr><tr><td> Bovine Serum Albumin </td><td></td><td></td></tr><tr><td> L-cystein </td><td></td><td></td></tr><tr><td> L-metionin </td><td></td><td></td></tr><tr><td> SDS (natrium dodecyl sulfate) </td><td></td><td></td></tr><tr><td> 2-mercaptoethanol </td><td></td><td></td></tr><tr><td> Glycine </td><td></td><td></td></tr><tr><td> Bromophenol blå </td><td></td><td></td></tr><tr><td> Cesium Klorid (CsCl) </td><td></td><td> &amp; Nbsp; </td></tr><tr><td> Kobber (I) Bromide (CuBr) </td><td></td><td></td></tr><tr><td> Kalsiumklorid (CaCl 2) </td><td></td><td></td></tr><tr><td> Kaliumklorid (KCl) </td><td></td><td></td></tr><tr><td> Magnesiumklorid (MgCl 2) </td><td></td><td></td></tr><tr><td> Natriumklorid (NaCl) </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> Alkyne probe for CuAAC * </td><td></td><td></td></tr><tr><td> Tamra Alkyne </td><td> Invitrogen </td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> Anstrengt alkyne probe for SPAAC * </td><td></td><td></td></tr><tr><td> Tamra DIBO Alkyne </td><td> Invitrogen </td><td> C10410 </td></tr></tbody></table> * Er Kjente leverandører av klikk kjemi reagenser og kits omfatter Invitrogen, Jena biovitenskap, Berry Associates, Sigma-Aldrich, Glen Forskning, Klikk kjemi Verktøy og Baseclick. En rekke alkyne fargestoffer og målretting ligander kan bli funnet på disse leverandørers kataloger.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

Modifikasjonen av viruspartikler har mottatt en betydelig mengde oppmerksomhet for sin enorme potensialet for å påvirke genterapi, oncolytic søknader og vaksineutvikling. 1,2,3 dagens tilnærminger til å endre viral overflater, som for det meste genetikk-baserte, ofte lider av demping virus produksjon, smittsomhet og cellulær transduksjon. 4,5 Bruke chemoselective klikk kjemi, har vi utviklet en grei alternativ tilnærming som sidesteps disse spørsmålene mens resterende både svært fleksibel og tilgjengelig. 1,2 Målet med denne protokollen er å demonstrere effekten av å bruke bioorthogonal klikk kjemi for å endre overflaten av adenovirus type 5 partikler. Denne to-trinns prosess kan brukes både terapeutisk 1 eller analytisk, 2,6 som det åpner for chemoselective ligation av målretting molekyler, fargestoffer eller andre molekyler av interesse på proteinerpre-merket med Azide tags. De tre store fordeler med denne metoden er at (1) metabolsk merking demonstrerer liten eller ingen innvirkning på viral fitness, 1,7 (2) et bredt spekter av effektor ligander kan utnyttes, og (3) det er bemerkelsesverdig rask, pålitelig og lett tilgang til. 1,2,7 I det første trinnet i denne fremgangsmåten, er adenovirus partikler produsert peiling enten azidohomoalanine (Aha, en metionin surrogat) eller unaturlig sukker O-forbundet N-azidoacetylglucosamine (O-GlcNAz), som begge inneholder natriumazid (-N 3) funksjonell gruppe. Etter rensing av azide-modifiserte viruspartikler, er en alkyne sonde som inneholder fluorescerende Tamra moiety ligated på en chemoselective måte til pre-merkede proteiner eller glykoproteiner. Til slutt, en SDS-PAGE-analyse utført for å demonstrere vellykkede ligation av sonden på de virale kapsid proteiner. Aha innlemmelse er vist å merke alle viral kapsidproteiner (Hexon, Penton og fiber), mens O-GlcNAz inkorporering resulterer i merking av Fiber bare. I dette utvikler feltet, har flere metoder for azide-alkyne ligation blitt utviklet, men bare de to vi har funnet å være mest praktisk er demonstrert her - stamme-forfremmet azide-alkyne cycloaddition (SPAAC) og kobber-katalysert azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosfære.

Watch this Scientific Journal Video about Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry at JoVE.com

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry (Video) | JoVE

Adenovirus partikler er konstruert for å inneholde enten unaturlig aminosyren analoge azidohomoalanine eller azido sukker<em&gt; O</em&gt;-GlcNAz. Den azide gruppe av hver er chemoselectively ligated via &quot;klikk&quot; kjemi reaksjoner som et middel for viral overflate modifikasjon.

Chemoselective Endring av Viral Overflater via Bioorthogonal Klikk kjemi

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic ...

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Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

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