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） 研究的目的是实现表面等离子体共振（SPR）成像方法， 研究蛋白质与DNA的相互作用。蛋白质的序列特异性结合 DNA在基因表达的调控中起着关键作用， 复制和重组。此外，识别和修饰 特定序列是生物DNA操作的关键组成部分， 修复系统。对这些蛋白质如何识别 某些寡核苷酸序列将有助于生物医学系统的设计 例如，它可以用来调节治疗基因的表达， proteins.因此，蛋白质-DNA相互作用的研究是一个快速发展的领域。 分子生物学的发展领域，部分得益于最近的进展， X射线结构测定方法。与此同时， 增加了从基因组中获得的可用基因组序列信息的量， 大规模的DNA测序工作产生了调查这一巨大数量的需求， 基因、操纵基因序列和其他蛋白质的新DNA序列数据 结合位点。为了支持这一努力，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， 蛋白质与大阵列双链DNA分子的结合 固定在化学修饰的金表面上。具体目标 项目将：（一）制造单链和双链的阵列 寡核苷酸序列与化学修饰的金表面，（ii） 证明了基于白色光的SPR成像系统对 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、外切核酸酶I和各种限制性内切核酸酶，（iv）监测 结合E.大肠杆菌双链 模拟野生型和突变的寡核苷酸序列， 二重对称lac算子位点，以及（v）研究序列 MutS蛋白结合的特异性（其参与了 甲基指导的DNA错配校正系统。大肠杆菌）至异源双链体 具有单碱基错配和1至4个碱基插入。