Here we explore the click chemistry-based synthesis of high affinity multivalent aptamers and their application for virus detection. In contrast to attempts based on rigid constructs, such as aptamer-modified nanoparticles or hybridization-connected constructs, we propose highly flexible “star-shaped” multivalent aptamers to facilitate the cooperative binding to randomly distributed viral proteins on the surface of pleomorphic viruses such as the respiratory syncytial virus (RSV). Polyethylene glycols (PEGs) with 4 azide terminated flexible arms spanning from a pentaerythritol core were conjugated to aptamer strands with dibenzocyclooctyne and a fluorescent label at their, 3’ and 5’ terminus, respectively. By controlling the aptamer - PEG ratio and following electrophoretic separation all the different valency aptamer conjugates could be prepared. For proof of concept, we used an aptamer selected for the G protein of RSV. We show that significantly higher apparent affinities can be obtained by using multivalent aptamers for both the G protein and RSV virus than with the single stranded aptamer, i.e. with up to ca. 2 orders of magnitude. The inherent convenience of synthesizing fluorescently labelled multivalent aptamers leads to a versatile class of affinity ligands for sensing as demonstrated through the improved detection sensitivity of tetravalent aptamer tagged RSV by fluorescence nanoparticle tracking analysis.

中文翻译：

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星形柔性臂多价适体可大幅提高病毒结合亲和力


在这里，我们探索基于点击化学的高亲和力多价适体的合成及其在病毒检测中的应用。与基于刚性结构的尝试相比，例如适配体修饰的纳米粒子或杂交连接的结构，我们提出了高度灵活的“星形”多价适配体，以促进与多形性病毒表面随机分布的病毒蛋白的协同结合，例如呼吸道合胞病毒（RSV）。具有 4 个叠氮化物末端柔性臂（从季戊四醇核心延伸）的聚乙二醇 (PEG) 与适体链缀合，适体链的 3' 和 5' 末端分别带有二苯并环辛炔和荧光标记。通过控制适体-PEG比例并随后进行电泳分离，可以制备所有不同价的适体缀合物。为了证明概念，我们使用了为 RSV G 蛋白选择的适体。我们表明，与使用单链适体相比，通过使用针对G蛋白和RSV病毒的多价适体可获得显着更高的表观亲和力，即高达约。 2个数量级。合成荧光标记的多价适体固有的便利性导致了用于传感的多功能亲和配体，通过荧光纳米颗粒跟踪分析提高了四价适体标记的 RSV 的检测灵敏度证明了这一点。