我们要感谢国家科学基金会拨款（CBET-0846259）。

请参阅表1，准备在这个协议中引用的所有媒体，缓冲区和解决方案。 1。生产的AHA-标记的腺病毒<ol><li>准备人类胚胎肾脏细胞11 100毫米组织培养皿（HEK 293）（ 见表1）在HEK 293细胞的生长介质保持在37°C，直到他们达到80 - 90％汇合。 （注:文化的汇合90％以上的感染，可能不会产生所需的病毒可能很难。） </li><li>松开的菜肴之一，首先消除的生长介质，5毫升的TD缓冲区冲洗一次，然后孵化一分钟1毫升的胰蛋白酶-EDTA（1×）的细胞的细胞。 </li><li>加入9毫升的HEK 293细胞的生长介质，并使用血球计数仪，指望从这个菜收获HEK 293细胞的数量。使用这个数字来计算金额感染腺病毒5型（Ad5的），使用1 multiplicit感染Y（MOI），价值10 PFU /细胞。账户的事实，Ad5的正常，传染性指数（传染性微粒的粒子总数的比例）通常是1:20。 </li><li>准备感染缓冲区10毫升。 </li><li>慢慢地从其余十培养皿HEK293细胞生长培养基，然后用5毫升的TD缓冲区。加入1毫升感染的缓冲区，以每道菜在37°C孵育1小时。在此期间感染期间，轻轻摇动每个培养皿一边到另一边，每隔15分钟。 </li><li>感染后在37°C，加入9毫升新鲜的HEK 293细胞生长培养基孵育18小时。 </li><li>编制阿哈标签介质（或符合控制介质控制）100毫升。务必过滤0.2微米的无菌注射器过滤器前准备的标签媒体集中的股票解决方案。 </li><li>仔细（以免洗去感染的细胞），取出后18 HR的HEK293细胞生长培养基第二洗每个培养皿中，用5毫升的TD缓冲区。 </li><li>删除TD缓冲洗涤液，每个培养皿中加入10毫升的阿哈标签介质。蛋氨酸控制介质控制，应在这一步，而不是的阿哈标签介质。 </li><li>标签介质在37°C为6小时的孵育感染的细胞。 ，随时标签可以在12 - 24小时后感染，但不应该是持续时间超过6小时的时间。此时间间隔已经确定，最大限度地与病毒结构蛋白翻译重叠，确保传染性不显着减少。7 </li><li>小心地取下标签介质，以免破坏细胞。新鲜HEK293细胞生长培养基中加入10毫升，每道菜，另外18小时在37°C孵育细胞。受感染的细胞在这一步，以尽量减少损失，这是完全没有必要删除标签解决方案。 HEK293细胞的培养基中含有过量的M等将outcompete任何剩余阿哈。 </li><li>继续加强净化3。 </li></ol> 2。生产的叠氮糖标记的腺病毒<ol><li>按照题为阿哈标记的腺病毒，包括步骤1.5生产过程。 </li><li>准备100毫升的叠氮糖标签介质。 </li><li>覆盖在37°C细胞10毫升新鲜叠氮糖标签介质和孵育44小时。 </li></ol> 3。叠氮化物标记的腺病毒颗粒的分离纯化<ol><li>从每个培养皿和锥形管转移到受感染的细胞取出媒体。在2000克离心10分钟，在4°C。取出上清液和悬浮颗粒在TD缓冲区（使用TD每10道菜的缓冲区8毫升）。 </li><li>重复（3个或更多）冻融循环使用杜瓦瓶液氮和37℃水浴裂解感染细胞。在离心2000克10分钟在4°C，从上清中分离出来的细胞碎片。 </li><li>准备先成超清晰的离心管中吸取2.5毫升1.25克/毫升的氯化铯溶液1 CsCl密度梯度离心管。接下来，使用其尖端定位在试管底部吸管慢慢加入2.0毫升1.4克/毫升的氯化铯溶液，小心不要打扰梯度界面。 </li><li> CsCl密度梯度管的顶部3.2从步骤上，吸取上清液，如果有必要附加的TD缓冲区，以填补管。摆动斗在125000克的转子为1小时15°在超高速离心Ç离心样品。 </li><li>超速完成后，应该是在氯化铯的两个解决方案的界面作为一个厚厚的白色条纹可见病毒颗粒。做一个解决漏滴用无菌针管底部的小穿孔。 </li><li>收集了厚厚的白色带，其中应包含标记Ad5的颗粒，羚牛Ğ照顾，以排除多余的氯化铯的解决方案。避免收集细胞蛋白，这将带病毒层以上。此外排除空病毒粒子衣壳形成了较轻的一层厚厚的白色病毒带略高于。 </li><li>另外，执行对Ad5的粒子标记的最终净化。加入收集的分数从3.6步超离心管4毫升和1.35克/毫升的氯化铯溶液的填充管。离心125000克为18小时，于15°C，并收集在中间的步骤3.5和3.6所述的管形成厚厚的白色带。 （注意:如果病毒滴度低，一条白色带可能不可见第二离心后，将不可能恢复标记的病毒）。 </li><li>对于存放过长时间的病毒粒子，所收集的病毒提取透析应进行对病毒的存储缓冲区。透析的病毒样本储存在-80°C </li></ol> 4。里使用应变促进叠氮炔环加成炔探头修饰的腺病毒颗粒gation（SPAAC） <ol><li>到50μL病毒的存储缓冲区的Ad5的改性叠的解决方案，增加0.25μlSPAAC标签解决方案的。 （请参阅市售紧张炔商贩名单试剂表）。 </li><li>允许应变推动的环加成反应，继续在室温下过夜。如果使用光敏感探头（ 如 :TAMRA），包括反应容器用铝箔。 </li><li>净化标记的病毒，根据步骤6。 </li></ol> 5。结扎，炔探头在无氧大气中使用的铜（I）催化叠氮，炔环加成（CuAAC）修饰的腺病毒颗粒<ol><li>地方7.2毫克的铜（I）溴化物（溴化亚铜）Eppendorf管中的粉末，用铝箔覆盖。 </li><li>吸取1毫升DMSO成为第二个Eppendorf管。 </li><li>准备标签的CuAAC溶液50μl的在第三个Eppendorf管中。用铝箔覆盖，如果炔探头是光敏感（ 如 :TAMRA-炔）。 </li><li> 6小时3个Eppendorf管，不封顶，将在无氧手套袋。所有样品应保持里面的手套袋，直到完成的CuAAC反应。 </li><li>准备加入1毫升的DMSO 7.2毫克溴化亚铜粉末，这两个已经按照步骤5.4无氧的溴化亚铜50×原液。 </li><li>启动此溴化亚铜原液加入1μl移液器50μL的CuAAC标签解决方案，并已按照步骤5.4无氧CuAAC反应。使反应进行12小时内手套袋。 </li><li>净化标记的病毒，根据步骤6。 </li></ol> 6。标记病毒颗粒的纯化和贮存<ol><li>净化标记的病毒样本，使用离心九月凝胶过滤自旋Çolumns平衡与病毒存储缓冲区，按照制造商的指引。 </li><li>另外，可以进行净化透析对病毒的存储缓冲区。 </li><li>应储存在-80°C标记的病毒颗粒</li></ol> 7。标记的腺病毒荧光凝胶扫描的SDS-PAGE <ol><li>纯化标记病毒，煮沸10分钟至20μL加入20μLLaemmli样品缓冲液（2×）。样品离心30秒，以消除任何不溶性的沉淀。如果残余铜是目前净化后，沸腾的样品可以导致SDS-PAGE凝胶上蛋白的粗条纹，导致在可视化的困难。在这种情况下，放弃在此步骤中样品的沸点。 </li><li>将凝胶电泳细胞的录像带，并添加运行缓冲。病毒的蛋白质样品，装入井所需的数量。使用一个加载前STAINED分子量标记。运行1小时凝胶在200五，用铝箔覆盖整个期间保持电泳细胞减少的漂白的荧光标签。 </li><li>为最佳分辨率，并删除所有无荧光，允许运行跟踪染料的凝胶电泳。停止电泳和快速传输凝胶荧光凝胶扫描仪扫描凝胶在适当波长（574纳米）为:TAMRA荧光发射。 </li><li>每个病毒颗粒结合的染料分子的数量可以通过测量荧光的几个标准:TAMRA浓度和样品比较，从步骤7.3量化。 </li></ol> 8。代表结果 SDS-PAGE电泳和荧光凝胶扫描共轭:TAMRA-Ad5的观测结果如图3所示。虽然这些荧光的凝胶扫描进行样品作案通过CuAAC田间，结果应该是比较时探头通过SPAAC结扎。这些凝胶扫描表明，在阿哈三个Ad5的衣壳蛋白（Hexon蛋白，聚氯，纤维）的修改标签的结果，而糖的标签修改只有光纤衣壳蛋白。此外，增加在阿哈浓度的（4毫米与32毫米）的结果更大程度在阿哈成立的，但它也已显示，以减少传染性。前7结果表明，在4mm生产Ad5的阿哈，大约有5％的暴露蛋氨酸网站的是:TAMRA修饰，这个数字提高到10％时使用32毫米阿哈是。这相当于约280和510，分别为每Ad5的粒子，染料标记网站。糖标签1的分析表明，大约22 36 Ad5的糖基化位点是由叠氮糖占领，随后:TAMRA标记。 如果10个组织培养皿用于生产叠改性Ad5的描述<html>在这个协议中，200 - 300μL的叠氮化改性病毒应获得3.6步骤中收集的白色带，以滴度约为1.5×10 12颗粒/毫升。此过程的一个共同困难是缺乏病毒带在此纯化步骤，这可以归因于病毒滴度低。这是一般是过高或过低合流，或洗去细胞的感染细胞的结果 - 成为从感染后板松动而在第1步 - 冲洗。 <img src="/files/ftp_upload/4246/4246fig1.jpg" alt="图1" /> 图1。协议概述。叠氮含AHA或GalNAz首次被纳入到腺病毒颗粒。结扎与炔的荧光探针，然后通过"点击"化学。最后，SDS-PAGE电泳用于证明结扎荧光探针。 igure 2"/&gt; 图2。炔探头结扎上叠（一）SPAAC，（二）CuAAC下，含氧及（c）含氧气氛中通过修改病毒。 <img src="/files/ftp_upload/4246/4246fig3.jpg" alt="图3" /> 图3。荧光标记叠，腺病毒5型后CuAAC:TAMRA-炔共轭（Ad5的）的SDS-PAGE扫描。 （一）AHA-Ad5的标记，使用两种浓度阿哈。标签似乎发生腺病毒Hexon，聚氯和光纤衣壳蛋白，与阿哈浓度增加增加标签的程度。 （二）AC 4 GalNAz-标记的腺病毒似乎只能在光纤衣壳蛋白标记。 <table border="1"><tbody><tr><td> 解 </td><td> 准备 </td><td> 笔记 </td></tr><tr><td> AD5存储缓冲区</td><td> 5毫米的Tris-HCl缓冲液（pH 8.0）中含有0.5毫米50 mM氯化钠， 氯化镁 ，25％（V / V）甘油，0.05％（W / V）牛血清白蛋白（BSA）的</td><td>储存在-20°C </td></tr><tr><td> AD5感染缓冲区</td><td> 2％（V / V）小牛血清，1×TC的解决方案，在存储缓冲区AD5（量确定使用MOI 10 PFU /细胞和传染性指数1:20）。使用TD缓冲区最终体积带来的解决方案</td><td> Ad5的浓度可通过紫外吸收光谱测定。 </td></tr><tr><td> TC方案（200×） </td><td> 136毫米100毫米氯化钙 ，氯化镁2 </td><td>保存于4°C间</td></tr><tr><td>运输署缓冲区</td><td> 137 mM氯化钠，氯化钾5毫米，0.07毫米的Na 2 HPO 4，和25毫米的Tris-HCl，pH值7.5 </td><td>保存于4°C间</td></tr><tr><td> HEK293细胞生长培养基</td><td>辅以10％（V / V），小牛血清（BCS）的1％（V / V）笔链球菌杜尔贝科的改良Eagle培养基（DMEM培养基） </td><td>保存于4°C间</td></tr><tr><td>半胱氨酸原液（100×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以200毫米L-半胱氨酸</td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>阿哈原液（25×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以100毫米L-Azidohomoalanine </td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>阿哈标签介质</td><td> DMEM培养基（-蛋氨酸/半胱氨酸），辅以10％BCS，阿哈储备溶液（1×），2毫米L-半胱氨酸</td><td>使用前准备好新鲜。 这相当于一个4毫米Concentration啊哈，虽然不同浓度可用于（见代表结果 ）。 </td></tr><tr><td>气象原液（25×） </td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以100毫米L-蛋氨酸</td><td>准备新鲜和过滤0.2微米的无菌注射器过滤器在使用前。 </td></tr><tr><td>气象控制介质</td><td> DMEM培养基（-蛋氨酸/半胱氨酸）辅以10％BCS的，符合储备溶液（1×），2毫米L-半胱氨酸</td><td>使用前准备好新鲜。 </td></tr><tr><td> AC 4 GalNAz，储备溶液（1000×） </td><td> 50毫米peracetylated的N - azidoacetylgalactosamine（AC 4 GalNAz）甲醇</td><td>保存于4°C。 AC 4 GalNAz是一个新陈代谢的前兆GlcNAz。 </td></tr><tr><td>叠氮糖标签介质</td><td> 100μLAC 4 GalNAz股票的解决方案，100毫升的HEK 293细胞生长培养基</td><td>允许甲醇蒸发之前加入HEK293细胞生长培养基。 </td></tr><tr><td>氯化铯解决方案</td><td>氯化铯溶解在TD缓冲区: 1.25克/毫升（18.08克/ 50毫升H 2 O） 1.35克/毫升（25.60克/ 50毫升H 2 O） 1.40克/毫升（31.00克/ 50毫升H 2 O） </td><td>密度梯度超速</td></tr><tr><td>病毒存储缓冲区</td><td>磷酸盐缓冲液（PBS），pH值7.2，含0.5毫米，0.9毫米氯化镁 ， 氯化钙 ，和10％（V / V）甘油</td><td>储存在-20°C </td></tr><tr><td> :TAMRA-BCN（SPAAC）的标签解决方案</td><td>在DMSO:TAMRA-BCN的: <ol><li>使用紫外线ABsorbance（外径260法）确定在步骤4.1滴度的病毒解决方案</li><li>在50μL等份确定的总数量（不适用Ad5的病毒颗粒） </li><li> :TAMRA-BCN的摩尔浓度（男）决定使用: çBCN的Ad5的 = 4×10 8×（不适用 /否AVO） 列印的AVO = 6.022×10 23 （在第4步准备，使反应混合物中包含100个病毒颗粒炔探头） </li></ol></td><td>应变推动炔探头的 对于SDS-PAGE电泳，约10 12颗粒/毫升的病毒滴度建议</td></tr><tr><td> :TAMRA-炔（CuAAC）的标签解决方案</td><td> 50μL叠改性Ad5的病毒存储缓冲区（Ad5的例确定为以上），1.5毫米batho<html>菲罗啉磺酸钠（ 即 1.5×最终溴化亚铜浓度）:TAMRA-炔。 :TAMRA-炔烃的摩尔浓度（米）的决定: çALK = 2×10 6×（N 重组腺病毒Ad5 / AVO） （在第5步准备，使反应混合物中包含100个病毒颗粒炔探头） </td><td> Tris缓冲被称为是一个有竞争力的铜和抑制配体8。 </td></tr><tr><td> Laemmli样品缓冲液（2×） </td><td> 125毫米的Tris-HCl（pH值6.8），4％（W / V）SDS，20％（V / V）甘油，10％（V / V）2 - 巯基乙醇，0.004％（W / V）溴酚蓝</td><td></td></tr><tr><td>运行缓冲液</td><td> 187毫米甘氨酸，3.5毫米，19毫米的Tris-HCl SDS H 2 O </td><td>保存于4°C间</td></tr></tbody></table>T" 表1。制备的缓冲区和本议定书所规定的解决方案。

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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>

没有利益冲突的声明。

在化学选择性和bioorthogonal点击反应，包括叠氮化合物的的发展是一个发展迅速的研究领域，以及随后出现的这些反应越来越多的选择，从生物耦合应用。我们已经限制了该协议的范围包括在我们自己的实验室和所有试剂的商业可用性的方法，因为它们的用途选择只有两个。 在第一个这样的路线-应变促进叠-炔环加成（SPAAC） -炔载内cyclooctyne环（ 图2a）。这是最近...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>腺病毒5型（Ad5的）含有绿色荧光蛋白转基因</td><td> BCBC </td><td> 391 </td></tr><tr><td>人类胚胎肾细​​胞（HEK 293） </td><td> ATCC </td><td> CRL-1573 </td></tr><tr><td>杜尔贝科的改良Eagle培养基（DMEM培养基），高糖</td><td> Invitrogen公司</td><td> 11965-092 </td></tr><tr><td>杜尔贝科的改良Eagle培养基，无蛋氨酸，没有半胱氨酸（DMEM-蛋氨酸/半胱氨酸），高糖</td><td> Invitrogen公司</td><td> 21013-024 </td></tr><tr><td>小牛血清</td><td> Invitrogen公司</td><td> 16170-078 </td></tr><tr><td>青霉素 - 链霉素100×解决方案（笔链球菌） </td><td> Invitrogen公司</td><td> 15140-122 </td></tr><tr><td> 0.5％胰蛋白酶-EDTA（10×） </td><td&gt; Invitrogen公司</td><td> 15400-054 </td></tr><tr><td> 100 MM细胞培养皿中，组织培养处理的聚苯乙烯</td><td>屋宇署猎鹰</td><td> 353003 </td></tr><tr><td>细胞培养二氧化碳培养箱</td><td></td><td></td></tr><tr><td>血球</td><td></td><td></td></tr><tr><td>生物安全柜</td><td></td><td></td></tr><tr><td>无菌注射器过滤器（0.2微米，醋酸纤维素） </td><td>厂商VWR </td><td> 28145-477（NA）514-0061（欧洲） </td></tr><tr><td>阿凡提乙脑离心机</td><td>贝克曼</td><td> 369001 </td></tr><tr><td>锥形管（50毫升，无菌） </td><td>屋宇署猎鹰</td><td> 352098 </td></tr><tr><td>管，薄壁，超清晰，13.2毫升，14 x 89毫米</td><td>贝克曼</td><td> 344059 </td></tr><tr><td>超速离心机配备与西南41和SW 60 Rotor </td><td>贝克曼</td><td></td></tr><tr><td> Eppendorf公司Biopur安全锁管，1.5毫升</td><td> Eppendorf公司</td><td> 0030 121.589 </td></tr><tr><td>离心机5418 </td><td> Eppendorf公司</td><td> 5418 </td></tr><tr><td>离心九月凝胶过滤离心柱</td><td>普林斯顿大学分离</td><td> CS-901 </td></tr><tr><td>无菌注射针，18针</td><td></td><td></td></tr><tr><td>氮手套袋（如果进行无氧CuAAC） </td><td></td><td></td></tr><tr><td>杜瓦瓶</td><td></td><td></td></tr><tr><td>液氮</td><td></td><td></td></tr><tr><td>电泳槽</td><td></td><td></td></tr><tr><td>荧光凝胶扫描仪</td><td></td><td></td></tr><tr><td>准备凝胶的Tris-HCl凝胶</td><td> BIO-RAD </td><td> 161-1105 </td></tr><tr><td> L - Azidohomoalanine </td><td> AnaSpec </td><td> 63669 </td></tr><tr><td>耶拿生物科学</td><td> CLK-AA005 </td><td></td></tr><tr><td> peracetylated 的 N-azidoacetylgalactosamine（AC 4 GalNAz） </td><td> Invitrogen公司</td><td> C33365 </td></tr><tr><td> Thermo Scientific的</td><td> 88905 </td><td></td></tr><tr><td> Sigma-Aldrich公司</td><td> A7480 </td><td></td></tr><tr><td> bathophenanthroline二磺酸（BDA）的钠盐</td><td> MP的Biomedicals </td><td> 0215011201 </td></tr><tr><td>二甲基亚砜（DMSO） </td><td></td><td></td></tr><tr><td>甲醇</td><td></td><td></td></tr><tr><td> Tris碱（三（羟甲基）氨基甲烷） </td><td></td><td></td></tr><tr><td>磷酸氢二钠（钠2 HPO 4） </td><td></td><td></td></tr><tr><td>磷酸盐缓冲液（PBS） </td><td></td><td></td></tr><tr><td> 1M盐酸</td><td></td><td></td></tr><tr><td>甘油</td><td></td><td></td></tr><tr><td>牛血清白蛋白</td><td></td><td></td></tr><tr><td> L-半胱氨酸</td><td></td><td></td></tr><tr><td> L-蛋氨酸</td><td></td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td></td><td></td></tr><tr><td> 2 - 巯基乙醇</td><td></td><td></td></tr><tr><td>甘氨酸</td><td></td><td></td></tr><tr><td>溴酚蓝</td><td></td><td></td></tr><tr><td>氯化铯（CsCl） </td><td></td><td> ＆NBSP; </td></tr><tr><td>铜（I）溴化物（溴化亚铜） </td><td></td><td></td></tr><tr><td>氯化钙（ 氯化钙 ） </td><td></td><td></td></tr><tr><td>氯化钾（KCl） </td><td></td><td></td></tr><tr><td>氯化镁（MgCl 2的 ） </td><td></td><td></td></tr><tr><td>氯化钠（NaCl） </td><td></td><td></td></tr><tr><td colspan="3"></td></tr><tr><td> 炔CuAAC *探头 </td><td></td><td></td></tr><tr><td> :TAMRA炔</td><td> Invitrogen公司</td><td> T10183 </td></tr><tr><td colspan="3"></td></tr><tr><td> 紧张炔探头SPAAC * </td><td></td><td></td></tr><tr><td> :TAMRA迪波炔</td><td> Invitrogen公司</td><td> C10410 </td></tr></tbody></table> *点击化学试剂和试剂盒的著名厂商包括Invitrogen公司，耶拿的生物科技公司，贝里联营公司，Sigma-Aldrich公司，格伦研究"化学工具，Baseclick。各种炔染料和靶向配体，可以发现在这些厂商的产品目录。

chemoselective modification of viral surfaces via bioorthogonal click chemistry

病毒颗粒的修改已收到的关注显着，其影响的基因治疗，溶瘤应用和疫苗开发的巨大潜力。1,2,3修改病毒的表面，其中大多是遗传学的基础，当前的方法往往受到衰减生产病毒，传染性和细胞转导。4,5使用化学选择性的点击化学，我们已经开发出一个简单的替代方法回避这些问题，同时保持高度灵活和方便。1,2 本议定书的目标是展示使用bioorthogonal点击化学修改的5型腺病毒颗粒表面的成效。可用于同时治疗1或分析，2,6这两个步骤的过程，作为靶向分子，染料或其他分子的化学选择性结扎允许到蛋白质预先标记叠标签。这种方法的三个主要优点是:（1）代谢标记表明几乎没有病毒健身的影响，1,7（2）广泛的效应配体可以利用，（3）它是非常快速，可靠和易于访问。1,2,7 在此过程的第一步，腺病毒颗粒生产轴承要么azidohomoalanine（AHA，蛋氨酸的替代）或不自然的糖O型联的 N-azidoacetylglucosamine（ 邻 GlcNAz），它们都含有叠氮化物（氮3）功能组。净化后的叠改性病毒颗粒，包含:TAMRA荧光基炔探头结扎预标记的蛋白质或糖蛋白在化学选择性的方式。最后，进行SDS-PAGE分析证明到病毒衣壳蛋白探头成功结扎。阿哈纳入标记所有病毒衣壳蛋白（Hexon蛋白，聚氯和光纤），而O型 GlcNAz只标签纤维掺入结果。 在这个不断发展的领域，已成功开发出多个叠 - 炔结扎方法，但是只有两个我们已经找到了，是最方便的证明本 - 促进应变叠氮化物 - 炔环加成（SPAAC）和铜催化的叠炔环加成（CuAAC）在无氧气氛下。

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

腺病毒颗粒设计，含有非天然氨基酸类似物azidohomoalanine或叠氮糖<em&gt;Ø</em&gt; 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。

通过Bioorthogonal点击化学化学选择性病毒表面改性

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

24.0K 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。

视频: Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

Microfluidic On-chip Capture-cycloaddition Reaction to Reversibly Immobilize Small Molecules or Multi-component Structures for Biosensor Applications

Methods for rapid surface immobilization of bioactive small molecules with control over orientation and immobilization density are highly desirable for biosensor and microarray applications. In this Study, we use a highly efficient covalent bioorthogonal [4+2] cycloaddition reaction between trans-cyclooctene (TCO) and 1,2,4,5-tetrazine (Tz) to enable the microfluidic immobilization of TCO/Tz-derivatized molecules. We monitor the process in real-time under continuous flow conditions using surface plasmon resonance (SPR). To enable reversible immobilization and extend the experimental range of the sensor surface, we combine a non-covalent antigen-antibody capture component with the cycloaddition reaction. By alternately presenting TCO or Tz moieties to the sensor surface, multiple capture-cycloaddition processes are now possible on one sensor surface for on-chip assembly and interaction studies of a variety of multi-component structures. We illustrate this method with two different immobilization experiments on a biosensor chip; a small molecule, AP1497 that binds FK506-binding protein 12 (FKBP12); and the same small molecule as part of an immobilized and in situ-functionalized nanoparticle.

We present a method for rapid, reversible immobilization of small molecules and functionalized nanoparticle assemblies for Surface Plasmon Resonance (SPR) studies, using sequential on-chip bioorthogonal cycloaddition chemistry and antibody-antigen capture.

微流控芯片上捕获环加成反应可逆地固定小分子或多组分结构的生物传感器应用

Methods  for rapid surface immobilization of bioactive small molecules with control over  orientation and immobilization density are highly desirable for ...

科研

化学

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

高效抗体标记由应变叠氮推广 - 炔环加成特定站点

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

生物化学

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

免疫与感染

Optimized Incorporation of Alkynyl Fatty Acid Analogs for the Detection of Fatty Acylated Proteins using Click Chemistry

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.

The study of fatty acylation has important implications in cellular protein interactions and diseases. Presented here is a modified protocol to improve click chemistry detection of fatty acylated proteins, which can be applied in various cell types and combined with other assays, including pulse-chase and mass spectrometry.

优化了炔基脂肪酸类似物的掺入，用于使用点击化学检测脂肪酰化蛋白

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in ...

Design, Synthesis, and Photochemical Properties of Clickable Caged Compounds

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural diversity of currently available caging groups and the difficulties in synthetic modification without sacrificing their photolysis efficiencies are obstacles to expanding the repertoire of caged compounds for live cell applications. As the chemical modification of coumarin-type photo-caging groups is a promising approach for the preparation of caged compounds with diverse physical and chemical properties, we report a method for the synthesis of clickable caged compounds that can be modified easily with various functional units via the copper(I)-catalyzed Huisgen cyclization. The modular platform molecule contains a (6-bromo-7-hydroxycoumarin-4-yl)methyl (Bhc) group as a photo-caging group, which exhibits a high photolysis efficiency compared to those of the conventional 2-nitrobenzyls. General procedures for the preparation of clickable caged compounds containing amines, alcohols, and carboxylates are presented. Additional properties such as the water solubility and cell targeting ability can be readily incorporated into clickable caged compounds. Furthermore, the physical and photochemical properties, including the photolysis quantum yield, were measured and were found to be superior to those of the corresponding Bhc caged compounds. The described protocol could therefore be considered a potential solution for the lack of structural diversity in the available caged compounds.

A protocol for the synthesis and measurement of the photochemical properties of modular caged compounds with clickable moieties is presented.

可点击笼体化合物的设计、合成和光化学特性

Caged compounds enable the photo-mediated manipulation of the cell physiology with high spatiotemporal resolution. However, the limited structural ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

生物工程

Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, genetic programmability, and biodegradability. While they&#39;re highly amenable to bioconjugation reactions for adding synthetic ligands onto their surface, the range in bioconjugation methodologies on these aqueous born capsids is relatively limited. To facilitate the direction of functional biomaterials research, non-traditional bioconjugation reactions must be considered. The reaction described in this protocol uses dibromomaleimides to introduce new functionality in the solvent exposed disulfide bonds of a VLP based upon Bacteriophage Q&#946;. Furthermore, the final product is fluorescent, which has the added benefit of generating a trackable in vitro probe using a commercially available filter set.

Here, we present a procedure to fluorescently functionalize the disulfides on Q&#946; VLP with dibromomaleimide. We describe Q&#946; expression and purification, the synthesis of dibromomaleimide-functionalized molecules, and the conjugation reaction between dibromomaleimide and Q&#946;. The resulting yellow fluorescent conjugated particle can be used as a fluorescence probe inside cells.

Dibromomaleimide 二硫化学制备共轭诱导荧光 PEGylated 病毒样粒子

The recent rise in virus-like particles (VLPs) in biomedical and materials research can be attributed to their ease of biosynthesis, discrete size, ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

生物学

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

核酸，蛋白质与甲基转移和辅酶类似物序列特异性标签

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...