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.
Glycosylation is implicated in cell signaling, trafficking, and development, and aberrant glycosylation has been shown to be a hallmark of many disease states. The results of the studies in this proposal would introduce a method to study glycosylation on a specific subset of cells within a system that contains many other cell types. The specific, covalent nature of the tagging would allow for relatively long time courses to be studied, so glycosylation changes over time could be investigated. For example, these results would allow the changes in glycosylation in tumor models to be studied inside an animal host. Using the reagents developed in this proposal can lead to a better understanding of the glycosylation-related underlying mechanisms of development and diseases.
