Aptamer-based Glycomics Tools

描述（申请人提供）：蛋白质糖基化在生物过程中起着非常重要的作用。然而，快速检测和区分这些修改并不是一个微不足道的问题。因此，开发能够快速检测糖基化模式的新工具对糖组学领域具有重要意义。在此应用中，我们建议开发一种新的平台方法，用于选择具有区分糖基化模式能力的基于dna的糖蛋白适体。所提出的方法是基于我们的中心假设，即将硼酸片段纳入DNA文库将允许适体选择过程倾向于糖基化识别，因为众所周知，硼酸片段与碳水化合物上常见的二醇和羟基之间存在强相互作用。在这个快速通道R41/R42申请的第一阶段，我们建议使用从健康供体收集的模型糖蛋白、前列腺特异性抗原（PSA）来检验所提出方法的可行性。在证明可行性的基础上，我们将在第二阶段使用来自癌症患者的PSA，并开发对不同亚型PSA具有高亲和力和特异性的适配体。我们还将研究人类绒毛膜促性腺激素（hCG），它有许多糖基化位点。糖基化的变化与癌症、唐氏综合症和妊娠健康有关。我们还建议开发一种夹心法和一种检测PSA和hCG糖基化异构体的试剂盒，以及一种大规模化学合成硼酸修饰适配体的方法。在资助期结束时，我们计划向市场推出几种产品，包括：(1)具有不同糖基化模式的PSA DNA适体和试剂盒；(2)各种硼酸修饰的TTPs，供研究实验室开发他们自己感兴趣的糖产物的硼酸修饰DNA适体。在拨款期结束时，我们还应准备好根据客户的需要定制糖产物的DNA适体选择，并进一步评估使用PSA适体作为fda批准的前列腺癌诊断的临床潜力。

DESCRIPTION (provided by applicant): Protein glycosylation plays very important roles in biological processes. However, detecting and differentiating such modifications rapidly is not a trivial issue. Therefore, developing novel tools that allow for the rapid detection of glycosylation patterns is of tremendous importance to the field of glycomics. In this application, we propose to develop a novel platform approach for the selection of DNA-based aptamers for glycoproteins with the ability to differentiate glycosylation patterns. The proposed approach is based on our central hypothesis that incorporation of the boronic acid moiety into a DNA library will allow the aptamer selection process to gravitate toward glycosylation recognition due to the well known strong interactions between the boronic acid moiety and diols and hydroxyl groups commonly found on carbohydrates. In Phase 1 of this fast-track R41/R42 application, we propose to examine the feasibility of the proposed method using a model glycoprotein, prostate-specific antigen (PSA) collected from healthy donors. Upon demonstration of feasibility, we will use PSA from cancer patients in Phase 2 and develop aptamers with high affinity and specificity for different isoforms of PSA. We will also work on human chorionic gonadotropin (hCG), which has many glycosylation sites. Variations in glycosylation have been correlated with cancer, Down syndrome, and the health of pregnancy. We also propose to develop a sandwich assay and a kit for examination of PSA and hCG glycosylation isoforms and a method for the large scale chemical synthesis of boronic acid-modified aptamers. At the end of the grant period, we plan to bring to market several products including (1) DNA aptamers and kits for PSA with different glycosylation patterns and (2) various boronic acid-modified TTPs for research labs to develop boronic acid-modified DNA aptamers for glycoproducts of their own interest. At the end of the grant period we should also be ready to do custom selections of DNA aptamers for glycoproducts based on customers' needed and be read to further evaluate the clinical potential of using PSA aptamers as a FDA-approved prostate cancer diagnostics. PUBLIC HEALTH RELEVANCE: The application aims to develop new methods for studying protein modifications that could be one day be used for detecting and diagnosing diseases such as cancer, Down syndrome, and failing pregnancy.