Aptamer-based Proteomic Analysis for Cancer Signatures

基于适体的癌症特征蛋白质组学分析

• 批准号: 7224541
• 负责人: Stephen Patrick Walton
• 金额: $ 19.66万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-27 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7224541
• 关键词: measurement; neoplasm /cancer; oligonucleotides; proteins; proteomics

DESCRIPTION (provided by applicant): Cells are comprised of many types of molecules, including small organic and inorganic molecules, carbohydrates, lipids, proteins, and nucleic acids. All of these molecules act in an integrated fashion at the molecular level to result in macroscopic cellular, tissue, and systemic phenotypes. Complete understanding of biological function would require accurate measurement of all of these molecules simultaneously. Vlicroarrays have made it possible to analyze nucleic acids at the genome-scale, but techniques for other cellular molecules currently lag behind. With proteins as another class of molecules prominent in cellular function and communication, intense effort is being invested toward the development and implementation of proteomic strategies for the analysis of clinical and research samples. Current proteomic strategies take many forms but can be grossly categorized as either array-based or not. Multiple array-based strategies nave been developed based on antibody recognition of the proteins of interest. The use of antibodies, however, brings with it issues of reproducible antibody production, target binding affinity, and denaturation. It is proposed to develop a strategy by which cancer signatures can be established by measuring protein concentrations in an array-based proteomic technology using aptamers, protein-binding RNA molecules. To ensure that the method can be expanded to parallel studies, each aptamer will be labeled with a molecular barcode. The protein concentration will then be obtained by measuring the concentration of the barcode on the aptamer that binds specifically to that protein. The method will be tested on a cell-culture model of Type 2 diabetes, providing valuable data for technique development as well as an improved understanding of the response of the liver to inflammatory stresses. Aptamers will be used for solution phase measurement of acute phase proteins as a preliminary demonstration of the possibility of future implementation of the strategy in a microarray format. The specific aims of the proposed work are to: i) generate molecular barcode-containing, structure-switching aptamers by in vitro selection and identify conserved sequence and structural features; ii) quantify the affinities and association kinetics of aptamertarget protein binding interactions; and iii) measure the acute phase protein expression profile of stimulated rat hepatocytes in cell- culture using the molecular barcode-containing aptamer strategy.

描述（由申请人提供）： 细胞由多种类型的分子组成，包括小的有机和无机分子，碳水化合物，脂质，蛋白质和核酸。所有这些分子在分子水平上以综合方式起作用，从而导致宏观细胞，组织和全身表型。完全了解生物功能将需要同时准确地测量所有这些分子。 Vlicroars使分析基因组规模的核酸成为可能，但是目前落后于其他细胞分子的技术。由于蛋白质是细胞功能和交流中突出的另一类分子，因此正在为开发和实施蛋白质组学策略而投入强烈的努力，以分析临床和研究样本。当前的蛋白质组学策略采用多种形式，但可以将基于阵列的或不明确地分类。基于对蛋白的抗体识别而开发了多种基于数组的策略。然而，抗体的使用带来了可再现抗体产生，靶结合亲和力和变性的问题。有人建议通过使用适体，蛋白质结合的RNA分子在基于阵列的蛋白质组学技术中测量蛋白质浓度来建立癌症特征。为了确保该方法可以扩展到平行研究，每个适体将用分子条形码标记。然后，将通过测量特异性与该蛋白质结合的适体上的条形码浓度来获得蛋白质浓度。该方法将在2型糖尿病的细胞培养模型上进行测试，为技术开发提供有价值的数据，以及对肝脏对炎症应激的反应的了解。适体将用于对急性相蛋白的溶液相测量，以此作为初步证明，以表明将未来以微阵列格式实施该策略的可能性。拟议的工作的具体目的是：i）通过体外选择并识别保守的序列和结构特征来产生含分子条形码的结构切换适体； ii）量化Aptamertarget蛋白结合相互作用的亲和力和关联动力学； iii）使用含分子条形码的适体策略来测量细胞培养中刺激的大鼠肝细胞的急性相蛋白表达谱。