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

描述（由申请人提供）：基于APT-SNAP阵列的心脏生物标志物糖基化状态的核酸适配体检测pi: Christopher L. Warren和Mary 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的见解。临床使用的大多数心脏生物标志物用于检测心力衰竭，心血管领域非常需要跨越心血管疾病谱系的生物标志物，从发展中的动脉粥样硬化到晚期疾病。临床接受的心力衰竭生物标志物BNP是通过糖基化修饰的，而糖基化形式的BNP在心力衰竭和慢性肾功能衰竭患者中占主导地位。然而，BNP的主要诊断方法并不能检测到糖基化形式。此外，准确检测多种生物标志物的可靠技术，特别是糖基化或其他修饰形式的生物标志物，是缺乏的。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 （Aptamer Specificity and Affinity for Proteins）微阵列，它可以显示数百万个DNA和RNA适配体，作为设计、发现和优化心脏病生物标志物高亲和力适配体的高通量原型设备。核酸适体由DNA或RNA组成，可以采用紧凑的三维结构，识别具有特殊特异性和高亲和力的靶标，使其成为心脏生物标志物的传感器。在本提案中，将在APT-SNAP阵列上识别六种主要心脏生物标志物的适配体作为原理证明。该提案的一个关键发明是一种合成高密度RNA适体阵列的新方法。将对鉴定出的心脏生物标志物适体在人血清中的检测限进行测试。APT-SNAP阵列将识别识别生物标志物特定残基上不同糖基化结构的适体，使用NT-proBNP作为模型生物标志物。为了实现这一目标，我们将合成一组NT-proBNP蛋白，每个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形式的适配体。为了扩大检测的普遍性，超越目前在许多临床检测中使用的抗体的限制，荧光蛋白染料将用于将阵列荧光强度与生物标志物浓度相关联。这一基于糖组学/蛋白质组学的建议的具体目的是：设计新型高密度DNA和RNA适配体微阵列，以识别心脏生物标志物的适配体。2. 开发一组适体来检测特定的糖基化形式的心脏生物标志物。最终目标是开发APT-SNAP作为生物标志物发现平台（I期），临床关联其他生物标志物（II期），并开发一种负担得起的年度护理点诊断测试（IIB期），用于数百种心脏生物标志物供医生使用。这些技术将影响准确诊断心脏病的能力，允许更好的治疗选择，改善患者护理，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.