Early detection helps to increase the survival rate in cancer patients. One way to achieve this is the detection and analysis of molecular signatures or biomarkers that have been correlated to cancer development and prognosis. Along this line, there is a need for the development of new technologies for the molecular analysis of various cancer markers. Such spirit is reflected in an RFA (RFA-CA-05-002) requesting applications on developing new """"""""detection technologies and sensors of cancer and the structures and molecules important in its development and diagnosis,"""""""" among other things. In response to this RFA, we propose this feasibility study of a new platform technology that can be used for the rapid construction of fluorescent sensors for glycoproteins. We focus on glycoproteins because numerous such proteins have been implicated in cancer development. This method is based upon (1) the power of systematic evolution of ligands by exponential enrichment method (SELEX) in search of optimal oligonucleotide aptamers that can afford high affinity and specificity recognition of the target analytes, (2) the unique ability of boronic acids to recognize diol structures present on the saccharide part of glycoproteins, (3) our own development of several fluorescent boronic acid compounds that show very significant fluorescence intensity changes (17- to 200-fold) upon saccharide or glycoprotein binding. We hope to build synergy between the SELEX approach and the unique recognition of glycoprotein by boronic acids in making DNA aptamer-based fluorescent sensors that (1) have high affinity and specificity for the target glycoprotein and (2) exhibit very significant fluorescence intensity changes upon binding. Specifically, the project intends to develop a method to prepare DNA aptamers modified with our fluorescent boronic acid reporter compounds.
The specific aims of the projects include (1) the synthesis of fluorescent boronic acid compounds that show great fluorescence changes upon binding to saccharides, (2) incorporation of the fluorescent boronic moieties into nucleotides, 3) using the SELEX approach for the selection of sensors with optimal specificity and affinity, 4) validation of the sensor binding with glycoproteins in solution. For this feasibility study (R21), we have selected prostate-specific antigen (PSA) as our model glycoprotein because of its importance in cancer diagnosis and the fact that glycosylation variations distinguish between physiological and pathological PSA isoforms. Such fluorescent sensors, if developed, offer the advantage of rapid and sensitive detection, the potential for high throughput screening, and low cost. Furthermore, the same technology, once developed, can also be used for the construction of fluorescent sensors for other cancer-related glycoproteins.
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