Rubino Frederick A, Oum Yoon Hyeun, Rajaram Lakshmi, Chu Yanjie, Carrico Isaac S
Department of Chemistry, Stony Brook University, USA.
J Vis Exp. 2012 Aug 19(66):e4246. doi: 10.3791/4246.
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. Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction. Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible. 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 therapeutically or analytically, 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, (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access. 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 - strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.
病毒颗粒的修饰因其在基因治疗、溶瘤应用和疫苗开发方面的巨大潜力而受到了广泛关注。目前修饰病毒表面的方法大多基于遗传学，常常会导致病毒产生、感染性和细胞转导能力的减弱。我们利用化学选择性点击化学开发了一种直接的替代方法，该方法既能避开这些问题，又保持了高度的灵活性和可操作性。本方案的目的是证明使用生物正交点击化学修饰5型腺病毒颗粒表面的有效性。这个两步过程可用于治疗或分析，因为它允许将靶向分子、染料或其他感兴趣的分子化学选择性连接到预先用叠氮标签标记的蛋白质上。该方法的三个主要优点是：（1）代谢标记对病毒适应性几乎没有影响；（2）可以使用多种效应配体；（3）该方法非常快速、可靠且易于操作。在本过程的第一步，生产携带叠氮高丙氨酸（Aha，一种甲硫氨酸替代物）或非天然糖O-连接的N-叠氮乙酰葡糖胺（O-GlcNAz）的腺病毒颗粒，这两种物质都含有叠氮（-N3）官能团。在纯化叠氮修饰的病毒颗粒后，将含有荧光TAMRA部分的炔烃探针以化学选择性方式连接到预先标记的蛋白质或糖蛋白上。最后，进行SDS-PAGE分析以证明探针成功连接到病毒衣壳蛋白上。结果表明，掺入Aha可标记所有病毒衣壳蛋白（六邻体、五邻体和纤突），而掺入O-GlcNAz仅导致纤突被标记。在这个不断发展的领域中，已经成功开发了多种叠氮-炔烃连接方法；然而，本文仅展示了我们发现的两种最方便的方法——在脱氧气氛下的应变促进叠氮-炔烃环加成（SPAAC）和铜催化的叠氮-炔烃环加成（CuAAC）。
