Covalent protein-oligonucleotide conjugates by copper-free click reaction.
Covalent protein-oligonucleotide conjugates by copper-free click reaction.
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DOI：
10.1016/j.bmc.2012.05.017
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发表时间：
2012-07-15
影响因子：
3.5
通讯作者：
Taton, T. Andrew
中科院分区：
文献类型：
--
作者：
Khatwani, Santoshkumar L.;Kang, Jun Sung;Mullen, Daniel G.;Hast, Michael A.;Beese, Lorena S.;Distefano, Mark D.;Taton, T. Andrew
关键词：
共价蛋白-寡脱氧核苷酸（蛋白-ODN）缀合物在许多生物学应用中是有用的，但是在温和条件下以显著的产率合成离散缀合物（其中两个组分之间的连接在蛋白和ODN两者中的限定位置处）可能是一个挑战。在这篇文章中，我们展示了一种策略，使用应变促进叠氮化物-炔[3+2]环加成（SPAAC，无铜“点击”反应）合成离散的蛋白质-ODN缀合物。叠氮化物功能化的蛋白质，通过酶促异戊烯化的C-末端CVIA标签与合成的azidoprenyl二磷酸，被“点击”到ODN，已被修改与应变二苯并环辛炔（DIBO-ODN）。将得到的蛋白质-ODN缀合物纯化，并通过尺寸排阻色谱和凝胶电泳进行表征。我们发现，SPAAC生物缀合反应的产率和反应时间与先前报道的铜催化的叠氮化物-炔[3+2]环加成（CuAAC）生物缀合反应相当，但不需要催化剂。使用表面结合的DIBO-ODN作为异双功能接头，使用相同的SPAAC化学将叠氮修饰的蛋白质修饰到表面上。蛋白质与ODN的无Cu点击生物缀合是Cu催化点击方法的简单且通用的替代方法。
Covalent protein-oligodeoxynucleotide (protein-ODN) conjugates are useful in a number of biological applications, but synthesizing discrete conjugates—where the connection between the two components is at a defined location in both the protein and the ODN—under mild conditions with significant yield can be a challenge. In this article, we demonstrate a strategy for synthesizing discrete protein-ODN conjugates using strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free “click” reaction). Azide-functionalized proteins, prepared by enzymatic prenylation of C-terminal CVIA tags with synthetic azidoprenyl diphosphates, were “clicked” to ODNs that had been modified with a strained dibenzocyclooctyne (DIBO-ODN). The resulting protein-ODN conjugates were purified and characterized by size-exclusion chromatography and gel electrophoresis. We find that the yields and reaction times of the SPAAC bioconjugation reactions are comparable to those previously reported for copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC) bioconjugation, but require no catalyst. The same SPAAC chemistry was used to immobilize azide-modified proteins onto surfaces, using surface-bound DIBO-ODN as a heterobifunctional linker. Cu-free click bioconjugation of proteins to ODNs is a simple and versatile alternative to Cu-catalyzed click methods.
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影响因子：
46.2
作者：
Jewett JC;Bertozzi CR
通讯作者：
Bertozzi CR
影响因子：
3.2
作者：
Beatty, Kimberly E.;Fisk, John D.;Smart, Brian P.;Lu, Ying Ying;Szychowski, Janek;Hangauer, Matthew J.;Baskin, Jeremy M.;Bertozzi, Carolyn R.;Tirrell, David A.
通讯作者：
Tirrell, David A.
影响因子：
56.9
作者：
COREY, DR;SCHULTZ, PG
通讯作者：
SCHULTZ, PG
影响因子：
16.6
作者：
Dommerholt, Jan;Schmidt, Samuel;Temming, Rinske;Hendriks, Linda J. A.;Rutjes, Floris P. J. T.;van Hest, Jan C. M.;Lefeber, Dirk J.;Friedl, Peter;van Delft, Floris L.
通讯作者：
van Delft, Floris L.
DOI：
10.1073/pnas.0707090104
发表时间：
2007-10-23
影响因子：
11.1
作者：
Baskin, Jeremy M.;Prescher, Jennifer A.;Bertozzi, Carolyn R.
通讯作者：
Bertozzi, Carolyn R.