OPTICAL PROBES OF DNA BENDING AND WRAPPING

DNA 弯曲和包裹的光学探针

• 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
• 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

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 确定DNA与表面的相互作用，通过检查吸附 小寡核苷酸到蛋白质大小（直径<200埃） 无机基质 该项目将探讨小的约束力 具有特定序列和构象的寡核苷酸（本质上 例如，弯曲）成胶体硫化镉（CdS），这种材料可以 可以制成各种尺寸（10 - 200埃直径）， 各种表面基团（阳离子，阴离子，疏水，亲水， 等）。这些胶体颗粒在受到照射时会发光， 发光对结合到膜上的吸附物的性质和量敏感。 粒子表面 因此，DNA与CdS的结合将从 作为添加的寡核苷酸的函数的发光的猝灭 浓度. 由于胶体的小尺寸（高曲率）， 粒子，这里提出的方法是方便地适合调查 预先存在的曲率对DNA结合亲和力的影响 分子到表面。 此外，表面衍生化与简单的 阳离子、小有机分子和肽可以影响 的DNA，并提供急需的洞察力的因素，决定 核酸的高电荷表面与不同的 表面。 建议的具体目标如下： 1)为了表征DNA序列、构象和长度对 它与蛋白质大小的颗粒的结合。 特别是DNA分子 将利用和比较具有和不具有预先存在的弯曲。 变化 在发光作为DNA浓度的函数将被用来提取 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)的影响 颗粒大小和功能对特定DNA大小的结合将 追究 颗粒的范围将在~ 10 - 200埃 3)结合的寡核苷酸与 OH-自由基和DNA酶足迹法将用于获得以下信息： DNA在曲面上的结构。 4)DNA的影响 结构，无论是损坏或携带序列错配，将进行评估， 调查这些因素在多大程度上可以发挥作用， DNA与胶体颗粒的相互作用。