Selection of Boronic Acid-modified Aptamers for Glycoproteins

描述（由申请人提供）：翻译后/共翻译修饰深刻地影响许多蛋白质的活性和命运。然而，快速检测和区分各种这样的修改并不是一个微不足道的问题。对于糖基化尤其如此，它可以通过使用不同的糖基化模式在单个位点上具有许多不同的修饰形式。糖基化后结构特征的多样性对糖蛋白中糖基化模式的快速检测和区分提出了挑战。该应用程序的长期目标是开发一种新的平台方法，用于选择基于dna的适体，以特异性识别糖蛋白的糖基化位点，从而允许糖基化模式的分化。我们方法的关键是将硼酸片段整合到DNA文库中，以使适体选择过程偏向糖基化识别。该设计利用了(1)众所周知的硼酸部分与碳水化合物上常见的二醇和羟基之间的强相互作用，(2)通过指数富集法（SELEX）寻找最佳寡核苷酸适配体的系统进化能力，可以提供对目标分析物的高亲和力和高特异性识别，(3)我们在硼酸和dna基适配体选择方面的丰富经验。(4) PI实验室中大量硼酸的可用性，这些硼酸在糖结合时改变荧光特性。在这个应用中，我们计划使用两种模型糖蛋白，前列腺特异性抗原和纤维蛋白原，来开发方法。一旦开发成功，该平台方法应普遍适用于开发其他糖蛋白以及具有生物学意义的糖脂和糖类的适体。为了实现这一应用的目标，我们计划追求以下具体目标：(1)设计和合成硼酸修饰的三磷酸胸苷并将其整合到DNA中；(2)选择硼酸修饰的前列腺特异性抗原和纤维蛋白原两种模型糖蛋白的dna适配体；(3)基于糖基化模式分析适体结合靶标的能力，并寻找结合所需的最小结构要求；(4)开发大规模合成硼酸修饰dna适配体所需的化学物质。公共卫生相关性：该应用程序旨在开发诊断和检测人类疾病的新方法，包括癌症，细菌感染等。

DESCRIPTION (provided by applicant): Post-/co-translational modifications profoundly affect the activities and fate of many proteins. However, detecting and differentiating various such modifications rapidly is not a trivial issue. This is especially true for glycosylation, which can have many different modification forms at a single site by the use of different glycosylation patterns. This diversity in structural features after glycosylation presents a challenge for quick detection and differentiation of glycosylation patterns in a glycoprotein. The long-term goal of this application is the development of a novel platform approach for the selection of DNA-based aptamers for the specific recognition of the glycosylation sites of a glycoprotein and therefore allowing for the differentiation of glycosylation patterns. The key to our methodology is the incorporation of the boronic acid moiety into a DNA library to bias the aptamer selection process toward glycosylation recognition. The design takes advantage of (1) the well known strong interactions between the boronic acid moiety and diols and hydroxyl groups commonly found on carbohydrates, (2) the power of Systematic Evolution of Ligands by Exponential Enrichment method (SELEX) in search of optimal oligonucleotide aptamers that can afford high affinity and high specificity recognition of the target analytes, (3) our extensive experience working with boronic acids and DNA-based aptamers selection, and (4) the availability of a large number of boronic acids in the PI's lab that change fluorescent properties upon sugar binding. In this application, we plan to use two model glycoproteins, prostate specific antigen and fibrinogen, to develop the methodology. Once developed, the platform methodology should be generally applicable to developing aptamers for other glycoproteins as well as glycolipids and saccharides of biological significance. To achieve the goals of this application, we plan to pursue the following specific aims: (1) Design and synthesis of boronic acid-modified thymidine triphosphate for incorporation into DNA; (2) Selection of boronic acid-modified DNA-based aptamers for two model glycoproteins: prostate specific antigen and fibrinogen; (3) Analysis of the aptamers' ability to bind their targets based on glycosylation patterns and search for minimal structural requirements for binding; (4) Developing the chemistry needed for large scale synthesis of boronic acid-modified DNA-aptamers. PUBLIC HEALTH RELEVANCE: The application aims to develop new methods for diagnosing and detecting human diseases including cancer, bacterial infection, etc.