Using aptamers to selectively deliver reagents for bioorthogonal tagging of azido

使用适体选择性地递送用于叠氮基生物正交标记的试剂

• 批准号: 8572969
• 负责人: Gabriela De Almeida
• 金额: $ 3.61万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-21 至 2014-09-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8572969
• 关键词: Acids; Amides; Amines; Animal Model; Animals; Antigen Targeting; Azides; Binding; Biological Markers; Cancer Model; Carbamates; Carboxylic Acids; Cell Culture Techniques; Cells; Chemicals; Chemistry; Complex; Cultured Cells; Development; Disease; Engineering; Ensure; Environment; Evaluation; Flow Cytometry; Fluorescence Microscopy; Glutamate Carboxypeptidase II; Goals; Jurkat Cells; LNCaP; Label; Lead; Libraries; Link; Malignant Neoplasms; Malignant neoplasm of prostate; Methodology; Methods; Microscopy; Modeling; Mus; Nature; Pattern; Population; Procedures; Process; Reagent; Reporter; Signal Transduction; Surface; Synthesis Chemistry; System; Techniques; Technology; Testing; Time; Xenograft Model; Xenograft procedure; aptamer; base; cancer cell; cell type; fluorophore; functional group; glycosylation; model development; public health relevance; sugar; trafficking; tumor

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.

描述（由申请人提供）：本项目的目的是以细胞选择性方式使用基于叠氮基糖的生物正交化学。叠氮糖谱分析的通常程序涉及用叠氮糖处理系统中的每个细胞。然后引入配备有标签的第二试剂，其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体。然而，系统中能够掺入叠氮糖的所有细胞都将被标记。在此，我建议使用配备有癌症生物标志物靶向适体的第二试剂来靶向和标记异质细胞群中所需的癌细胞亚群。在目标1中，我描述了与sgc8c连接的二级试剂库的合成和初步评价，sgc8c是一种结合PTK7的适体，PTK7是各种癌细胞上的生物标志物。合成将涉及使已知试剂包括羧酸或酸等价物柄。柄将用于与在相对端上已经配备有标签的胺修饰的适体形成酰胺或氨基甲酸酯键。将在含叠氮糖的培养细胞上测试不同浓度的这些试剂，以找到仅允许标记表达PTK7的那些细胞的最佳试剂浓度组合。在目标2中，sgc8c部分将与另一种适体A10交换，以确保该技术不仅特异于sgc8c。然后将在混合培养细胞群中测试新试剂，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，这些模型已被代谢工程改造为包含叠氮糖，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。总之，我建议开发使用适体的试剂，使叠氮基糖为基础的生物正交化学细胞类型选择性。如果在小鼠癌症模型中取得成功，这种技术的选择性和共价性质可以帮助阐明癌细胞中随着时间的推移发生的糖基化模式的变化，并允许研究比目前可能的更多相关环境。