Aptamer-based Proteomic Analysis for Cancer Signatures

描述(由申请人提供)： 细胞由许多类型的分子组成，包括有机和无机小分子、碳水化合物、脂类、蛋白质和核酸。所有这些分子在分子水平上以一种综合的方式作用，导致宏观的细胞、组织和系统表型。完全了解生物功能需要同时准确地测量所有这些分子。超微阵列已经使在基因组规模上分析核酸成为可能，但其他细胞分子的技术目前落后了。随着蛋白质作为另一类分子在细胞功能和通讯中的突出地位，人们正投入大量精力开发和实施用于临床和研究样本分析的蛋白质组学策略。目前的蛋白质组策略有多种形式，但大体上可以分为基于阵列的策略和不基于阵列的策略。基于抗体对感兴趣蛋白质的识别，已经开发了多种基于阵列的策略。然而，抗体的使用带来了可重复产生的抗体、靶标结合亲和力和变性等问题。有人建议开发一种策略，通过使用适体，即蛋白质结合的RNA分子，在基于阵列的蛋白质组技术中测量蛋白质浓度，从而建立癌症特征。为了确保该方法可以扩展到平行研究，每个适配子都将被标记一个分子条形码。然后通过测量与该蛋白质特异结合的适体上条形码的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，为技术开发以及更好地了解肝脏对炎症应激的反应提供有价值的数据。适配子将用于急性相蛋白质的溶液相测量，作为未来以微阵列形式实施该战略的可能性的初步示范。这项工作的具体目的是：i)通过体外选择产生含有分子条形码的结构转换适配子，并确定保守的序列和结构特征；ii)量化适配子与靶蛋白结合作用的亲和力和结合动力学；以及iii)使用含有分子条形码的适配子策略测量细胞培养中刺激的大鼠肝细胞的急性期蛋白质表达谱。

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.