Using aptamers to selectively deliver reagents for bioorthogonal tagging of azido

描述（由申请人提供）：该项目的目标是以细胞选择性的方式使用叠氮糖为基础的生物正交化学。叠氮糖谱分析的通常程序包括用叠氮糖处理系统中的每个细胞。然后引入带有标签的二级试剂，它与糖上的叠氮化物官能团反应，在细胞上的糖和标签之间形成共价连接体。然而，系统中所有能够结合叠氮糖的细胞都将被标记。在此，我建议使用配备癌症生物标志物靶向适配体的二级试剂来靶向和标记异质细胞群中所需的癌细胞子集。在Aim 1中，我描述了与sgc8c连接的二级试剂库的合成和初步评估，sgc8c是一种结合PTK7的适配体，PTK7是各种癌细胞的生物标志物。该合成将涉及使已知试剂包含羧酸或酸当量柄。手柄将用于与胺修饰的适体建立酰胺或氨基甲酸酯连接，该适体已经在另一端配备了标签。这些试剂的不同浓度将在含叠氮糖的培养细胞上进行测试，以找到一个最佳的试剂浓度组合，只允许那些表达PTK7的细胞被标记。在Aim 2中，sgc8c部分将与另一个适配体A10交换，以确保该技术不仅针对sgc8c。然后，新试剂将在培养细胞的混合群体中进行测试，并使用显微镜验证选择性。最后，新试剂将在小鼠癌症模型中进行测试，这些模型经过代谢工程改造，加入了叠氮糖，以确保即使在这种复杂的环境中，也只有目标癌细胞被试剂标记。综上所述，我建议开发利用适体使叠氮糖基生物正交化学具有细胞类型选择性的试剂。如果在小鼠癌症模型中取得成功，该技术的选择性和共价特性可以帮助阐明癌细胞中糖基化模式随着时间的推移而发生的变化，并允许研究比目前可能的更多相关环境。

DESCRIPTION (provided by applicant): The objective of this project is to use azidosugar-based bioorthogonal chemistries in a cell-selective manner. The usual procedure for azidosugar profiling involves treating every cell in a system with azidosugar. A secondary reagent equipped with a tag is then introduced and it reacts with the azide functional group on the sugars, forming a covalent linker between the sugar on the cell and the tag. However, all of the cells in the system capable of incorporating azidosugar will be tagged. Herein, I propose using secondary reagents equipped with cancer biomarker-targeting aptamers to target and tag only a desired subset of cancer cells within a heterogeneous cell population. In Aim 1, I describe the synthesis and preliminary evaluation of a library of secondary reagents linked to sgc8c, an aptamer that binds PTK7, a biomarker on various cancer cells. The syntheses will involve making the known reagents to include a carboxylic acid or acid equivalent handle. The handle will be used to make an amide or carbamate linkage to the amine-modified aptamer that is already equipped with a tag on the opposite end. Different concentrations of these reagents will be tested on azidosugar-bearing cultured cells to find an optimal reagent-concentration combination that allows only those cells that express PTK7 to be labeled. In Aim 2, the sgc8c portion will be exchanged with another aptamer, A10, to ensure that the technology is not only specific to sgc8c. The new reagents will then be tested in mixed populations of cultured cells and selectivity will be verifie using microscopy. Finally, the new reagents will be tested in mouse cancer models that have been metabolically engineered to incorporate azidosugar to ensure that even in this complex setting, only the target cancer cells are labeled by the reagent. In summary, I propose to develop reagents that use aptamers to make azidosugar based bioorthogonal chemistries cell-type selective. If successful in mouse cancer models, the selective and covalent nature of this technology can help elucidate changes in glycosylation patterns that occur over time in cancer cells and allow more relevant environments to be studied than are currently possible.