Aptamer-Based Detection of Cardiac Biomarker Glycosylation States Using APT-SNAP Arrays PIs: Christopher L. Warren and Mary S. Ozers The presence of cardiac-related biomarkers in patient biospecimens can provide important insight into diagnosis of cardiovascular disease, its progression, and optimal therapies for treatment. Most of the cardiac biomarkers in clinical use detect heart failure, and the cardiovascular field is in great need of biomarkers that span the cardiovascular disease spectrum from developing atherosclerosis to late-stage disease. The clinically-accepted biomarker for heart failure, BNP, is modified by glycosylation, and the glycosylated form of BNP predominates in patients with heart failure and chronic renal failure. However, the leading diagnostic assay for BNP does not detect the glycosylated forms. Furthermore, robust technologies to accurately detect multiple biomarkers, especially glycosylated or other modified forms of biomarkers, are lacking. To address this significant diagnostic need for heart disease patients, we are developing the APT-SNAP (Aptamer Specificity and Affinity for Proteins) microarray, which displays millions of DNA and RNA aptamers, as a prototype high throughput device to design, discover, and optimize high affinity aptamers for heart disease biomarkers. Nucleic acid aptamers are composed of either DNA or RNA and can adopt a compact three-dimensional structure that recognizes a target with exceptional specificity and high affinity, making them useful as a sensor of cardiac biomarkers. In this proposal, aptamers will be identified on the APT-SNAP array for six major cardiac biomarkers as proof of principle. A key invention of the proposal is a novel methodology to synthesize high density RNA aptamers arrays. Identified aptamers for the cardiac biomarkers will be tested for their limit of detection n human serum. The APT-SNAP array will identify aptamers that recognize distinct glycosylation structures at specific residues of a biomarker, using NT-proBNP as a model biomarker. To accomplish this, we will synthesize a set of NT-proBNP proteins, each with a distinct glycosylation pattern, and iteratively design aptamers that specifically recognize the glycosylated BNP forms. To expand the universality of the assay beyond the limitations of antibodies currently used in many clinical assays, a fluorescent protein dye will be used to correlate array fluorescence intensity to biomarker concentration. The Specific Aims of this glycomics/proteomics-based proposal are: 1. Design novel high density DNA and RNA aptamer microarrays to identify aptamers for cardiac biomarkers. 2. Develop a panel of aptamers to detect specific glycosylated forms of cardiac biomarkers. The ultimate goals are to develop APT-SNAP as a biomarker discovery platform (Phase I), clinically correlate additional biomarkers (Phase II), and develop an affordable annual point-of-care diagnostic test (Phase IIB) for hundreds of cardiac biomarkers for physician use. These technologies will impact the ability to diagnose heart disease accurately, allowing for better treatment options, improved patient care, and longer lifespans.