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、脂质、蛋白质和核酸。所有这些分子在分子水平上以整合的方式起作用，从而产生宏观细胞、组织和全身表型。要完全了解生物功能，就需要同时精确测量所有这些分子。微阵列使得在基因组水平上分析核酸成为可能，但其他细胞分子的技术目前还落后。随着蛋白质作为另一类在细胞功能和通信中突出的分子，正在投入大量的努力来开发和实施用于临床和研究样品分析的蛋白质组学策略。目前的蛋白质组学策略有多种形式，但可以大致分为基于阵列或不基于阵列。基于抗体对目标蛋白质的识别，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再现的抗体产生、靶结合亲和力和变性的问题。建议开发一种策略，通过该策略可以通过使用适体（蛋白质结合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。然后通过测量特异性结合该蛋白质的适体上的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，为技术开发提供有价值的数据，以及更好地了解肝脏对炎症应激的反应。适体将被用于溶液相测量的急性期蛋白作为一个初步的示范，未来的战略实施的可能性，在一个微阵列格式。所提出的工作的具体目标是：i）通过体外选择产生含有分子条形码的结构转换适体，并鉴定保守序列和结构特征; ii）量化适体与靶蛋白结合相互作用的亲和力和缔合动力学;以及iii）使用含有分子条形码的适体策略测量细胞培养物中刺激的大鼠肝细胞的急性期蛋白表达谱。

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.