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.
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.
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