Aptamer-Based Detection of Cardiac Biomarker Glycosylation States Using APT-SNAP

描述（由申请人提供）：使用APT-SNAP阵列PI进行基于适体的心脏生物标志物糖基化状态检测：Christopher L.沃伦和玛丽S. Ozers患者生物标本中心脏相关生物标志物的存在可以为心血管疾病的诊断、进展和最佳治疗提供重要见解。临床使用的大多数心脏生物标志物检测心力衰竭，并且心血管领域非常需要跨越从发展动脉粥样硬化到晚期疾病的心血管疾病谱的生物标志物。临床上接受的心力衰竭生物标志物BNP通过糖基化修饰，并且BNP的糖基化形式在心力衰竭和慢性肾衰竭患者中占主导地位。然而，BNP的主要诊断测定不能检测糖基化形式。此外，缺乏准确检测多种生物标志物，特别是糖基化或其他修饰形式的生物标志物的稳健技术。为了满足心脏病患者的这一重要诊断需求，我们正在开发APT-SNAP（蛋白质适体特异性和亲和力）微阵列，它显示数百万个DNA和RNA适体，作为原型高通量设备，用于设计，发现和优化心脏病生物标志物的高亲和力适体。核酸适体由DNA或RNA组成，并且可以采用紧凑的三维结构，该结构以异常的特异性和高亲和力识别靶标，使其可用作心脏生物标志物的传感器。在该提案中，将在APT-SNAP阵列上识别六种主要心脏生物标志物的适体作为原理证明。该提案的一个关键发明是合成高密度RNA适体阵列的新方法。将检测已鉴定的心脏生物标志物适体在人血清中的检出限。APT-SNAP阵列将使用NT-proBNP作为模型生物标志物来鉴定识别生物标志物的特定残基处的不同糖基化结构的适体。为了实现这一目标，我们将合成一组NT-proBNP蛋白，每种蛋白都具有不同的糖基化模式，并迭代设计特异性识别糖基化BNP形式的适体。为了将测定的通用性扩展到目前用于许多临床测定的抗体的限制之外，将使用荧光蛋白染料将阵列荧光强度与生物标志物浓度相关联。这个基于糖组学/蛋白质组学的建议的具体目的是：1。设计新型高密度DNA和RNA适体微阵列，以鉴定心脏生物标志物的适体。2.开发一组适体以检测心脏生物标志物的特定糖基化形式。最终目标是开发APT-SNAP作为生物标志物发现平台（I期），临床相关的其他生物标志物（II期），并为数百种心脏生物标志物开发负担得起的年度床旁诊断测试（IIB期）供医生使用。这些技术将影响准确诊断心脏病的能力，从而提供更好的治疗选择，改善患者护理和延长寿命。

DESCRIPTION (provided by applicant): 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.