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） 研究是在实施表面等离子体共振（SPR）成像方法 蛋白-DNA相互作用的研究。蛋白质与 DNA在基因表达的调节和控制中起关键作用， 复制和重组。另外，识别和修改的酶 特定序列是生物DNA操纵和 维修系统。对这些蛋白质如何识别的增强理解 某些寡核苷酸序列将有助于设计生物医学系统 例如，可以用来调节治疗的表达 蛋白质。因此，蛋白-DNA相互作用的研究是一种迅速 分子生物学的生长区域，部分在NMR和NMR的最新进展中有助于 X射线结构测定方法。同时，爆炸性 从获得的可用基因组序列信息的量增加 大规模的DNA测序工作创造了需要调查的大量 基因，算子序列和其他蛋白质的新DNA序列数据 绑定位点。为了支持这项工作，我们的目标是使用SPR成像 技术是一种快速有效的方法，用于筛选序列特异性 蛋白质与大阵列的双链DNA分子的结合 固定在化学修饰的金表面上。特定目标 项目将是：（i）捏造单链和双链的阵列 化学修饰的金表面的寡核苷酸序列（ii） 证明白色光基SPR成像系统对 原位监测DNA杂交吸附和蛋白质吸附，（III） 通过T4 DNA监测表面结合的寡核苷酸的酶促操作 连接酶，外切核酸酶I和各种限制性核酸内切酶，（iv）监视 大肠杆菌的LAC阻遏蛋白与双链阵列的结合 寡核苷酸序列，该序列对野生型和突变进行建模 两倍对称的LAC操作员位点，（V）研究序列 蛋白质MUT的结合的特异性（涉及 大肠杆菌的甲基定向的DNA不匹配校正系统至异形电子 单基碱不匹配和1至4个基础插入。