Photogenerated Carbohydrate Microarrays

描述（由申请人提供）：碳水化合物识别在许多生物和疾病过程中起着核心作用。蛋白质-碳水化合物识别也具有新兴的医学重要性，例如在对抗感染、控制肿瘤扩散和靶向药物的开发中。糖基化微阵列是快速检测蛋白质和细胞糖基化模式的重要工具之一。目前，碳水化合物微阵列仍处于早期开发阶段，部分受到复杂制备方案的阻碍。在碳水化合物化学水平以及表面化学水平上，都需要新的、简单且稳健的阵列制造方法。本申请集中于设计和开发一种全新的微阵列化学，该微阵列化学是快速的并且容易被外部光调制。在阵列制造中使用的光致连接技术是通用的，可以容纳各种碳水化合物结构，并且与现有的微阵列技术高度兼容。在这项工作中，特别强调将放在控制配体显示方面的结合亲和力和特异性。此外，将进行溶液结合研究，以进一步测试阵列化学和所创建阵列的可靠性，以生成值得信赖的筛选结果。提出了四个具体目标。在目标1中，将开发两种一般方法用于将碳水化合物结构偶联到固体基质：即，光探针-碳水化合物缀合物的固定化，以及碳水化合物在光探针官能化表面上的直接偶联。这些阵列化学将适应从简单单糖到更复杂的糖缀合物的广泛的碳水化合物。在目标2中，将研究配体展示（配体呈递、接头长度、基质）对结合亲和力和特异性的影响。将开发条件以建立所生成的微阵列的再现性、稳定性和灵敏度。目标3的重点是验证溶液结合研究的光生微阵列的性能。蓝细菌凝集素Cyanovirin-N是一种HIV灭活蛋白，将用作微阵列验证和优化的基准系统。在目标4中，将展示所制备的微阵列在碳水化合物结合蛋白分析和高通量配体筛选中的用途。具体而言，优化的微阵列将用于筛选抗病毒药物。 公共卫生相关性：该项目的重点是开发碳水化合物微阵列，用于快速分析大量目标物种和高通量筛选方案。碳水化合物介导的识别在许多疾病过程中起着核心作用，例如细菌粘附到宿主组织和炎症过程。蛋白质-碳水化合物识别也具有新兴的医学重要性，例如在对抗感染、控制肿瘤扩散和靶向药物的开发中。

DESCRIPTION (provided by applicant): Carbohydrate recognition plays a central role in many biological and disease processes. Protein-carbohydrate recognition is also of emerging medical importance, for example in combating infection, controlling the spread of tumors, and the development of targeting drugs. Carbohydrate microarrays have been identified as one of the most important tools for rapid probing of functional properties of complex glycosylation patterns of proteins and cells. At present, carbohydrate microarrays are still in the early development phase, in part hampered by complex preparation protocols. New, simple and robust methods to array fabrication are needed, both on the carbohydrate chemistry level, as well as the surface chemistry level. The present application focuses on the design and development of a fundamentally new microarray chemistry that is fast and is easily modulated by external light. The photoligation technique used in the array fabrication is versatile, can accommodate a variety of carbohydrate structures, and is highly compatible with existing microarray technologies. In this work, special emphasis will be placed on the control of ligand display with respect to binding affinity and specificity. In addition, solution binding studies will be undertaken to further test the array chemistry and reliability of the created arrays in order to generate trustworthy screening results. Four Specific Aims are presented. In Aim 1, two general approaches will be developed for coupling carbohydrate structures to solid substrates: namely, immobilization of photoprobe- carbohydrate conjugates, and direct coupling of carbohydrates on photoprobe-functionalized surfaces. These array chemistries will accommodate a wide range of carbohydrates from simple monosaccharides to more complex glycoconjugates. In Aim 2, the impact of ligand display (ligand presentation, linker length, matrix) on binding affinity and specificity will be studied. Conditions will be developed to establish reproducibility, stability, and sensitivity of generated microarrays. Aim 3 focuses on validating the performance of photogenerated microarrays with solution binding studies. The cyanobacterial lectin Cyanovirin-N, an HIV-inactivating protein, will be used as the benchmark system for the validation and optimization of microarrays. In Aim 4, the use of the prepared microarrays in carbohydrate-binding protein analysis and high- throughput ligand screening will be demonstrated. Specifically, the optimized microarrays will be employed for screening anti-viral agents. PUBLIC HEALTH RELEVANCE: This project focuses on developing carbohydrate microarrays for the rapid analysis of large volumes of target species and high-throughput screening protocols. Carbohydrate-mediated recognition plays a central role in many diseases processes, for instance bacterial adherence to host tissue and inflammatory processes. Protein-carbohydrate recognition is also of emerging medical importance, for example in combating infection, controlling the spread of tumors, and the development of targeting drugs.