SPR Imaging Studies of DNA and Protein Microarrays

DNA 和蛋白质微阵列的 SPR 成像研究

• 批准号: 6613554
• 负责人: ROBERT M CORN
• 金额: $ 25.36万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613554
• 关键词: DNA binding protein; Escherichia coli; binding sites; biological signal transduction; endopeptidases; enzyme activity; gene expression; glutathione transferase; gold; high throughput technology; imaging /visualization /scanning; maltose; microarray technology; phosphotransferases; polymerase chain reaction; protein binding; protein protein interaction; proteomics; surface plasmon resonance; technology /technique development

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.

描述（由申请人提供）：这项研究的总体目的是开发表面等离子体共振（SPR）成像的标签免费检测方法，用于以阵列格式的生物障碍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异性结合相互作用将以SPR成像测量为特征。这项研究的很大一部分将集中在化学修饰的金表面上生物分子的特征良好，高度可重现，可重现，健壮和生物活性阵列的制造。该项目将要研究的特定生物系统是：（i）细菌响应调节蛋白与DNA阵列的序列特异性结合，以帮助确定细菌如何通过两个成分信号转导系统和基因表达对外部刺激响应，以及对这些蛋白酶的结合（II）的结合（II）的结合（II）的结合（II）稳健和定向的肽阵列可帮助确​​定该肽特异性如何用于控制细胞过程，以及（iii）可以在蛋白质组学研究中实现的定向融合蛋白阵列的设计。定向融合蛋白阵列的制造将需要以阵列格式的捕获剂（例如谷胱甘肽或麦芽糖）附着在化学修饰的金表面上，然后将融合蛋白的结合（包括谷胱甘肽-S-转移酶（GST）或麦芽糖结合蛋白（MBP）包含在内。 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的定向，以使溶液中的其他蛋白可以访问它们。通常，以阵列形式研究生物疗法相互作用的SPR成像技术的开发将有助于加速搜索和发现方法，这些方法正成为现代生物学研究的越来越重要的工具。