SPR Imaging Studies of DNA and Protein Microarrays

描述（由申请人提供）：本研究的总体目标是开发表面等离子体共振（SPR）成像的无标签检测方法，用于阵列格式的生物亲和相互作用的高通量研究。蛋白质与DNA、肽或蛋白质的生物分子阵列的特定结合相互作用将用SPR成像测量来表征。这项研究的一个重要部分将集中于在化学修饰的金表面上制造具有良好特征、高度可重复性、健壮性和生物活性的生物分子阵列。本项目将研究的具体生物系统有：(1)细菌反应调节蛋白与DNA阵列的序列特异性结合，以帮助确定细菌如何通过双组分信号转导系统和基因表达对外部刺激作出反应，并筛选抑制这些蛋白结合的化合物文库；(2)激酶的序列特异性酶活性；磷酸酶和蛋白酶具有强大的定向肽阵列，以帮助确定如何使用这种肽特异性来控制细胞过程，以及（iii）定向融合蛋白阵列的设计，可以在蛋白质组学研究中实施。定向融合蛋白阵列的制造需要将捕获剂（如谷胱甘肽或麦芽糖）以阵列形式附着在化学修饰的金表面上，然后将融合蛋白(包括谷胱甘肽s转移酶（GST）或麦芽糖结合蛋白（MBP）结合到阵列元素上。GST或MBP与表面捕获剂的特定相互作用有助于确保融合蛋白以这样一种方式定向，即它们将被溶液中的其他蛋白质所接近。总的来说，用于研究阵列形式的生物亲和相互作用的SPR成像技术的发展将有助于加速搜索和发现方法，这些方法正日益成为现代生物学研究的重要工具。

DESCRIPTION (provided by applicant): The overall aim of this research is the development of the label free detection method of surface plasmon resonance (SPR) imaging for the high-throughput study of bioaffinity interactions in an array format. The specific binding interactions of proteins with biomolecular arrays of DNA, peptides, or proteins will be characterized with SPR imaging measurements. A significant portion of this research will focus on the fabrication of well-characterized, highly reproducible, robust, and bioactive arrays of biomolecules on chemically modified gold surfaces. The specific biological systems that will be studied in this project are: (i) the sequence specific binding of bacterial response regulator proteins to DNA arrays in order to help determine how bacteria respond to external stimuli via two component signal transduction systems and gene expression, and to screen libraries of compounds that inhibit the binding of these proteins, (ii) the sequence specific enzymatic activity of kinases, phosphatases and proteases with robust and oriented peptide arrays to help determine how this peptide specificity is used to control cellular processes, and (iii) the design of oriented fusion protein arrays that can be implemented in proteomics studies. The fabrication of oriented fusion protein arrays will require the attachment of capture agents such as glutathione or maltose onto chemically modified gold surfaces in an array format, followed by the binding of fusion proteins that include either glutathione-s-transferase (GST) or maltose binding protein (MBP) onto the array elements. The specific interaction of the GST or MBP with the surface capture agent helps insure that the fusion proteins are oriented in such a manner that they will be accessible to other proteins in solution. In general, the development of SPR imaging techniques for the study of bioaffinity interactions in an array format will help accelerate the search and discovery approaches that are becoming increasingly essential tools of modern biological research.