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。叠氮多糖图谱的通常程序包括用叠氮多糖处理系统中的每个细胞。然后引入配备标签的二次试剂，它与糖上的叠氮官能团反应，在细胞上的糖和标签之间形成共价连接物。然而，系统中所有能够掺入叠氮葡萄糖的细胞都将被标记。在此，我建议使用配备癌症生物标记物靶向适体的二级试剂来靶向和标记异质细胞群体中所需的癌细胞子集。在目标1中，我描述了与sgc8c相连的二级试剂文库的合成和初步评估，sgc8c是一种适体，可以结合各种癌细胞上的生物标记物PTK7。这些合成将包括使已知的试剂包括羧酸或酸当量的手柄。手柄将被用来使酰胺或氨基甲酸酯与胺修饰的适配子相连，该适配子的另一端已经配备了标签。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，以找到最佳的试剂浓度组合，仅允许标记那些表达PTK7的细胞。在目标2中，sgc8c部分将与另一适配子A10交换，以确保该技术不仅适用于sgc8c。然后，新试剂将在混合培养细胞群体中进行测试，并将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，加入了叠氮葡萄糖，以确保即使在这种复杂的环境中，也只有靶癌细胞被试剂标记。综上所述，我建议开发使用适配子的试剂，使基于叠氮糖的生物正交化学具有细胞类型选择性。如果在小鼠癌症模型中取得成功，这项技术的选择性和共价性可以帮助阐明癌细胞中随时间发生的糖基化模式的变化，并允许比目前可能的更相关的环境进行研究。

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.