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.
The application aims to develop new methods for diagnosing and detecting human diseases including cancer, bacterial infection, etc. ? ? ?
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