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。对于糖基化来说尤其如此，通过使用不同的糖基化模式，糖基化可以在单个位置具有多种不同的修饰形式。糖基化后结构特征的这种多样性给快速检测和区分糖蛋白中的糖基化模式带来了挑战。这一应用的长期目标是开发一种新的平台方法来选择基于DNA的适体，用于特定识别糖蛋白的糖基化位点，从而允许糖基化模式的差异化。我们方法学的关键是将硼酸部分整合到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烈相互作用，(2)通过指数富集法(SELEX)系统进化配体的能力，以寻找能够对目标分析物提供高亲和力和高特异性识别的最佳寡核苷酸适配子，(3)我们在硼酸和DNA适体选择方面的丰富经验，以及(4)PI实验室提供的大量可以在糖结合时改变荧光性质的硼酸。在这项应用中，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，以发展方法学。一旦开发出来，平台方法应该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的适配子。为了实现这一应用的目标，我们计划追求以下具体目标：(1)设计和合成用于掺入DNA的硼酸修饰的胸苷三磷酸；(2)针对两种模型糖蛋白：前列腺特异性抗原和纤维蛋白原选择基于硼酸修饰的DNA适配子；(3)基于糖基化模式分析适配子结合其靶点的能力，并寻找结合所需的最低结构要求；(4)发展大规模合成硼酸修饰的DNA适配子所需的化学条件。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.