We would like to acknowledge the NSF for funding (CBET- 0846259).

Refer to Table 1 for preparation of all media, buffers and solutions referenced in this protocol.
1. Production of Aha-Labeled Adenovirus
<ol>
 <li> Prepare eleven 100-mm tissue culture dishes of human embryonic kidney cells (HEK 293) maintained in HEK 293 cell growth medium (see Table 1) at 37 &deg;C until they have reached 80 - 90% confluency. (Note: Cultures with confluency over 90% may be difficult to infect and may not produce viruses as desired.) <...

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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>

The development of chemoselective and bioorthogonal click reactions, including those of azides, is a rapidly-evolving area of research, and subsequently there is a growing number of these reactions to choose from for bioconjugation applications. We have restricted the scope of this protocol to include only two methods which were chosen because of their usefulness in our own lab and commercial availability of all reagents.
In the first such route - strain-promoted azide-alkyne cycloaddition (SP...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Adenovirus type 5 (Ad5) containing a GFP transgene</td>
 <td>BCBC</td>
 <td>391</td>
 </tr>
 <tr>
 <td>Human Embryonic Kidney (HEK 293) cells</td>
 <td>ATCC</td>
 <td>CRL-1573</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium (DMEM), High Glucose</td>
 <td>Invitrogen</td>
 <td>11965-092</td>
 </tr>
 <tr>
 <td>Dulbecco's Modified Eagle Medium, no Methionine, no Cysteine (DMEM -Met / -Cys), High Glucose</td>
 <td>Invitrogen</td>
 <td>21013-024</td>
 </tr>
 <tr>
 <td>Bovine Calf Serum</td>
 <td>Invitrogen</td>
 <td>16170-078</td>
 </tr>
 <tr>
 <td>Penicillin - Streptomycin 100&times; Solution (Pen Strep)</td>
 <td>Invitrogen</td>
 <td>15140-122</td>
 </tr>
 <tr>
 <td>0.5% Trypsin-EDTA (10&times;)</td>
 <td>Invitrogen</td>
 <td>15400-054</td>
 </tr>
 <tr>
 <td>100 mm Cell Culture Dish, tissue-culture treated polystyrene</td>
 <td>BD Falcon</td>
 <td>353003</td>
 </tr>
 <tr>
 <td>Cell culture CO2 incubator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hemocytometer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biosafety cabinet</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sterile syringe filter (0.2 &mu;m, cellulose acetate) </td>
 <td>VWR</td>
 <td>28145-477 (NA), 514-0061 (Europe)</td>
 </tr>
 <tr>
 <td>Avanti J-E centrifuge</td>
 <td>Beckman Coulter</td>
 <td>369001</td>
 </tr>
 <tr>
 <td>Conical tubes (50 ml, sterile)</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>Ultracentrifuge equipped with an SW 41 and SW 60 rotor</td>
 <td>Beckman</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Eppendorf Biopur Safe-Lock Tubes, 1.5 ml</td>
 <td>Eppendorf</td>
 <td>0030 121.589</td>
 </tr>
 <tr>
 <td>Centrifuge 5418</td>
 <td>Eppendorf</td>
 <td>5418</td>
 </tr>
 <tr>
 <td>Centri-Sep gel filtration spin columns</td>
 <td>Princeton Separations</td>
 <td>CS-901</td>
 </tr>
 <tr>
 <td>Sterile needle, 18 gauge</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nitrogen glove bag (if deoxygenated CuAAC is to be performed)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dewar flask</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Liquid nitrogen</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrophoresis cell</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluorescent gel scanner</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ready 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 Biosciences</td>
 <td>CLK-AA005</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz)</td>
 <td>Invitrogen</td>
 <td>C33365</td>
 </tr>
 <tr>
 <td>Thermo Scientific</td>
 <td>88905</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sigma-Aldrich</td>
 <td>A7480</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bathophenanthroline disulfonic acid (BDA) disodium salt</td>
 <td>MP Biomedicals</td>
 <td>0215011201</td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Methanol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tris base (tris(hydroxymethyl)aminomethane)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Disodium Phosphate (Na2HPO4)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1M HCl</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bovine Serum Albumin</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-cysteine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-methionine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SDS (sodium dodecyl sulfate)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-mercaptoethanol</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycine</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Bromophenol blue</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cesium Chloride (CsCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Copper(I) Bromide (CuBr)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Calcium Chloride (CaCl2)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium Chloride (KCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Magnesium Chloride (MgCl2)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Chloride (NaCl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td colspan="3">&nbsp;</td>
 </tr>
 <tr>
 <td>Alkyne probe for CuAAC*</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TAMRA Alkyne</td>
 <td>Invitrogen</td>
 <td>T10183</td>
 </tr>
 <tr>
 <td colspan="3">&nbsp;</td>
 </tr>
 <tr>
 <td>Strained alkyne probe for SPAAC*</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TAMRA DIBO Alkyne</td>
 <td>Invitrogen</td>
 <td>C10410</td>
 </tr>
</tbody>
</table>
* Notable vendors of click chemistry reagents and kits include Invitrogen, Jena Biosciences, Berry Associates, Sigma-Aldrich, Glen Research, Click Chemistry Tools, and Baseclick. A variety of alkyne dyes and targeting ligands can be found in these vendors' catalogs.

chemoselective modification of viral surfaces via bioorthogonal click chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.1,2
The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically1 or analytically,2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.1,2,7
In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N3) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.
In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein &#x2013; strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.

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

Adenovirus particles are engineered to contain either the unnatural amino acid analogue azidohomoalanine or the azido sugar O-GlcNAz. The azide group of each is chemoselectively ligated via "click" chemistry reactions as a means of viral surface modification.

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

chemoselective-modification-viral-surfaces-via-bioorthogonal-click

Research

JoVE Journal

Immunology and Infection

23.7K 次观看.  Stony Brook University. 腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖Ø GlcNAz。每叠组chemoselectively结扎通过“点击”化学反应病毒表面改性的一种手段。