OPTICAL PROBES OF DNA BENDING AND WRAPPING

DNA 弯曲和包裹的光学探针

基本信息

批准号：
2024121
负责人：
Catherine J. MURPHY
金额：
$ 7.28万
依托单位：
UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-05-01 至 2002-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2024121
关键词：
DNA DNA footprinting cadmium chemical binding conformation gel mobility shift assay luminescence nucleic acid sequence particle surface property

项目摘要

DESCRIPTION: This is a proposal designed to understand the factors that determine the interaction of DNA with surfaces, by examining the adsorption of small oligonucleotides to protein-sized (< 200Angstrom in diameter) inorganic substrates. The project will explore the binding of small oligonucleotides with specific sequences and conformations (intrinsically bent, for example) to colloidal cadmium sulfide (CdS), a material that can be made in a variety of sizes (10 - 200 Angstrom diameter) and with a variety of surface groups (cationic, anionic, hydrophobic, hydrophilic, etc.). These colloidal particles emit luminescence when irradiated, and this luminescence is sensitive to the nature and amount of adsorbate bound to the particle surface. Thus, binding of the DNA to CdS will be obtained from the quenching of the luminescence, as a function of added oligonucleotide concentration. Because of the small size (high curvature) of the colloidal particles, the method proposed here is conveniently suited to investigate the effect of pre-existing curvature on the binding affinity of the DNA molecules to the surface. Moreover, surface derivatization with simple cations, small organic molecules, and peptides can influence the binding of the DNA and provide much needed insight into the factors that determine the interaction of the highly charged surface of nucleic acids with different surfaces. The following specific aims are proposed: 1) To characterize the effect of DNA sequence, conformation and length on its binding to the protein-sized particles. In particular, DNA molecules with and without pre-existing bends will be utilized and compared. Changes in luminescence as a function of DNA concentration will be used to extract binding constants. Longer in-phased curved DNAs will be used to determine the influence of curvature on the surface binding. 2) The effect of particle size and functionality on the binding of a particular DNA size will be investigated. The range of the particles will vary in the range of ~ 10 - 200Angstrom. 3) Accessibility of the bound oligo nucleotides to OH-radical-and DNase footprinting will be used to obtain information about the structure of the DNA on the curved surfaces. 4) The effect of DNA structures, either damaged or carrying sequence mismatches, will be assessed to investigate to what degree these factors can play a role in the interaction of the DNA with the colloidal particles.

描述：这是一项旨在了解影响因素的提案通过检查吸附来确定 DNA 与表面的相互作用小寡核苷酸到蛋白质大小（直径 < 200 埃）无机基材。该项目将探索小型的绑定具有特定序列和构象的寡核苷酸（本质上例如，弯曲）到胶体硫化镉（CdS），这是一种可以可制成各种尺寸（直径 10 - 200 埃）并具有多种表面基团（阳离子、阴离子、疏水性、亲水性、 ETC。）。这些胶体颗粒在受到照射时会发出冷光，这发光对与吸附物结合的吸附物的性质和量敏感颗粒表面。因此，DNA 与 CdS 的结合将从发光淬灭，作为添加寡核苷酸的函数专注。由于胶体尺寸小（曲率高）粒子，这里提出的方法很适合研究预先存在的曲率对 DNA 结合亲和力的影响分子到表面。此外，表面衍生化具有简单阳离子、小有机分子和肽可以影响结合 DNA 并提供急需的关于决定因素的见解核酸的高电荷表面与不同的相互作用表面。提出以下具体目标： 1) 表征DNA序列、构象和长度对DNA的影响它与蛋白质大小的颗粒结合。特别是DNA分子将利用和比较有和没有预先存在的弯曲。变化发光作为 DNA 浓度的函数将用于提取结合常数。更长的同相弯曲 DNA 将用于确定曲率对表面结合的影响。 2）效果颗粒大小和功能对特定 DNA 大小的结合被调查。粒子的范围将在〜10的范围内变化 - 200埃。 3) 结合寡核苷酸的可及性 OH-自由基和 DNase 足迹将用于获取有关弯曲表面上的 DNA 结构。 4）DNA的作用将评估受损或带有序列不匹配的结构调查这些因素在多大程度上发挥作用 DNA 与胶体颗粒的相互作用。