SPR IMAGING STUDIES OF PROTEIN/DNA INTERACTIONS

蛋白质/DNA 相互作用的 SPR 成像研究

• 批准号: 2881586
• 负责人: ROBERT M CORN
• 金额: $ 25.24万
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2881586
• 关键词: DNA; DNA binding protein; Escherichia coli; adsorption; bioengineering /biomedical engineering; bioimaging /biomedical imaging; exonuclease; gold; intermolecular interaction; nucleic acid hybridization; phosphoester ligase; protein binding; restriction endonucleases; surface coating; surface plasmon resonance; technology /technique development

DESCRIPTION: (adapted from applicant's abstract) The overall aim of this research is to implement surface plasmon resonance (SPR) imaging methods in the study of protein-DNA interactions. The sequence-specific binding of proteins to DNA plays a pivotal role in the regulation and control of gene expression, replication and recombination. In addition, enzymes that recognize and modify specific sequences are critical components of biological DNA manipulation and repair systems. An enhanced understanding of how these proteins recognize certain oligonucleotide sequences would aid in the design of biomedical systems which could, for example, be used to regulate the expression of therapeutic proteins. For this reason, the study of protein-DNA interactions is a rapidly growing area of molecular biology, aided in part by recent advances in NMR and X-ray structural determination methods. At the same time, the explosive increase in the amount of available genomic sequence information obtained from large scale DNA sequencing efforts creates the need to survey this vast amount of new DNA sequence data for genes, operator sequences and other protein binding sites. In support of this effort, our goal is to use SPR imaging techniques as a rapid and efficient method for screening the sequence-specific binding of proteins to large arrays of double-stranded DNA molecules immobilized at chemically modified gold surfaces. Specific goals of this project will be to: (i) fabricate arrays of single- and double-stranded oligonucleotide sequences to chemically modified gold surfaces, (ii) demonstrate the sensitivity of a white light-based SPR imaging system to monitor in situ both DNA hybridization adsorption and protein adsorption, (iii) monitor the enzymatic manipulation of surface-bound oligonucleotides by T4 DNA ligase, Exonuclease I, and various restriction endonucleases, (iv) monitor the binding of the lac repressor protein of E. coli to arrays of double-stranded oligonucleotide sequences which model both the wild type and mutations of the two-fold symmetric lac operator site, and (v) investigate the sequence specificity of the binding of the protein MutS (which is involved in the methyl-directed DNA mismatch correction system of E. coli) to heteroduplexes with single base mismatches and 1 to 4 base insertions.

描述：（改编自申请人的摘要）本项目的总体目标 研究的目的是应用表面等离子共振（SPR）成像方法 研究蛋白质-DNA 相互作用。蛋白质的序列特异性结合 DNA在基因表达的调节和控制中发挥着关键作用， 复制和重组。此外，识别和修饰的酶 特定序列是生物 DNA 操作的关键组成部分 修复系统。加深对这些蛋白质如何识别的理解 某些寡核苷酸序列将有助于生物医学系统的设计 例如，它可以用于调节治疗性药物的表达 蛋白质。因此，蛋白质-DNA相互作用的研究迅速发展。 分子生物学领域的不断发展，部分得益于核磁共振和核磁共振的最新进展 X射线结构测定方法。与此同时，爆炸性的 获得的可用基因组序列信息量的增加 大规模 DNA 测序工作需要对如此大量的数据进行调查 基因、操纵子序列和其他蛋白质的新 DNA 序列数据 结合位点。为了支持这项工作，我们的目标是使用 SPR 成像 技术作为筛选序列特异性的快速有效的方法 蛋白质与大量双链 DNA 分子的结合 固定在经过化学修饰的金表面。本次具体目标 项目将：（i）制造单链和双链阵列 化学修饰金表面的寡核苷酸序列，(ii) 展示基于白光的 SPR 成像系统的灵敏度 原位监测 DNA 杂交吸附和蛋白质吸附，(iii) 监测 T4 DNA 对表面结合寡核苷酸的酶促操作 连接酶、核酸外切酶 I 和各种限制性核酸内切酶，(iv) 监测 大肠杆菌的 lac 阻遏蛋白与双链阵列的结合 模拟野生型和突变型的寡核苷酸序列 双重对称 lac 操作位点，以及 (v) 研究序列 蛋白质 MutS 结合的特异性（参与 大肠杆菌的甲基定向 DNA 错配校正系统）到异源双链体 具有单碱基错配和 1 至 4 个碱基插入。