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: 7089832
• 负责人: ROBERT M CORN
• 金额: $ 27.5万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 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 2007-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7089832
• 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）：本研究的总体目标是开发表面等离子共振（SPR）成像的无标记检测方法，用于阵列形式的生物亲和相互作用的高通量研究。蛋白质与 DNA、肽或蛋白质生物分子阵列的特异性结合相互作用将通过 SPR 成像测量来表征。这项研究的很大一部分将集中于在化学修饰的金表面上制造特征良好、高度可重复、稳健且具有生物活性的生物分子阵列。该项目将研究的具体生物系统是：(i) 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两部分信号转导系统和基因表达对外部刺激作出反应，以及筛选抑制这些蛋白质结合的化合物文库，(ii) 使用稳健且定向的肽阵列确定激酶、磷酸酶和蛋白酶的序列特异性酶活性，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) ） 这设计可在蛋白质组学研究中实施的定向融合蛋白阵列。定向融合蛋白阵列的制造需要将谷胱甘肽或麦芽糖等捕获剂以阵列形式附着到化学修饰的金表面上，然后结合包括谷胱甘肽-S-转移酶 (GST) 或麦芽糖结合在内的融合蛋白蛋白质（MBP）到阵列元素上。 GST 或 MBP 与表面捕获剂的特异性相互作用有助于确保融合蛋白以可被溶液中的其他蛋白接触的方式定向。总的来说，用于以阵列形式研究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，这些方法正成为现代生物学研究日益重要的工具。