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：Christopher L.Warren和Mary S.Ozers基于适体的心脏生物标记物糖基化状态的检测患者生物标本中与心脏相关的生物标记物的存在可以为心血管疾病的诊断、其进展和最佳治疗方法提供重要的见解。临床上使用的大多数心脏生物标志物都能检测到心力衰竭，心血管领域迫切需要跨越从动脉粥样硬化到晚期疾病的心血管疾病范围的生物标志物。临床公认的心力衰竭生物标志物BNP是通过糖基化修饰的，在心力衰竭和慢性肾功能衰竭患者中，BNP的糖基化形式占主导地位。然而，对BNP的领先诊断分析不能检测到糖基化形式。此外，缺乏准确检测多个生物标记物的可靠技术，特别是糖基化或其他修饰形式的生物标记物。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(蛋白质适配子特异性和亲和力)微阵列，它显示了数百万个DNA和RNA适配子，作为设计、发现和优化心脏病生物标志物的高亲和力适配子的原型高通量设备。核酸适配子由DNA或RNA组成，可以采用紧凑的三维结构，以特殊的特异性和高亲和力识别靶标，使它们成为心脏生物标志物的传感器。在这项提案中，将在APT-SNAP阵列上为六个主要的心脏生物标志物确定适配子作为原则证据。该方案的一个关键发明是一种合成高密度RNA适配子阵列的新方法。已确定的心脏生物标志物适配子将在人血清中进行检测限度测试。APT-SNAP阵列将识别识别生物标记物特定残基上不同糖基化结构的适体，使用NT-proBNP作为模型生物标记物。为了实现这一点，我们将合成一组NT-proBNP蛋白质，每个蛋白质都有不同的糖基化模式，并反复设计特异性识别糖基化BNP形式的适体。为了扩大分析的广泛性，超越目前在许多临床分析中使用的抗体的限制，将使用荧光蛋白染料将阵列荧光强度与生物标记物浓度相关联。这个基于糖组/蛋白质组学的提案的具体目的是：1.设计新型高密度DNA和RNA适配子微阵列来识别心脏生物标志物的适配子。2.开发一组适配子来检测特定糖基化形式的心脏生物标志物。最终目标是开发APT-SNAP作为生物标记物发现平台(第一阶段)，在临床上关联更多的生物标记物(第二阶段)，并为数百种供医生使用的心脏生物标记物开发负担得起的年度护理点诊断测试(第二阶段B)。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、更好的患者护理和更长的寿命。

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.