OPTICAL PROBES OF DNA BENDING AND WRAPPING

DNA 弯曲和包裹的光学探针

• 批准号: 2701808
• 负责人: Catherine J. MURPHY
• 金额: $ 13.22万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 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/2701808
• 关键词: DNA; DNA footprinting; cadmium; chemical binding; conformation; gel mobility shift assay; luminescence; nucleic acid sequence; particle; surface property

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：这是一项旨在理解以下因素的提案 通过检测表面的吸附来确定DNA与表面的相互作用 从小分子寡核苷酸到蛋白质大小(直径200埃) 无机底物。该项目将探索具有约束力的小型 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)，一种可以 可制成各种尺寸(直径10-200埃)，并具有 各种表面基团(阳离子、阴离子、疏水、亲水、 等)。这些胶体粒子在受到照射时会发出发光，这 发光是敏感的性质和数量的吸附结合到 粒子曲面。因此，DNA与CD的结合将从 作为添加的寡核苷酸的函数的发光猝灭 集中精神。由于胶体的尺寸小(高曲率) 颗粒，本文提出的方法方便地适用于研究 预先存在的曲率对DNA结合亲和力的影响 分子到达表面。此外，用简单的表面衍生法 阳离子、有机小分子和多肽可以影响结合 DNA，并提供了对决定 高电荷态核酸表面与不同分子间的相互作用 表面。提出了以下具体目标： 1)研究DNA序列、构象和长度对DNA序列的影响 它与蛋白质大小的颗粒结合在一起。尤其是DNA分子 使用和不使用预先存在的折弯将被利用和比较。变化 在发光中作为DNA浓度的函数将被用来提取 结合常数。将使用更长的同相弯曲DNA来确定 曲率对表面结合的影响。2)的影响 颗粒大小和官能度对特定DNA大小的结合 被调查。粒子的射程将在~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DNA酶足迹将用于获取以下信息 DNA在曲面上的结构。4)DNA的作用 将评估损坏或携带序列不匹配的结构 为了调查这些因素在多大程度上可以在 DNA与胶体颗粒的相互作用。