Nanoscale DNA Diagnostics by Single Molecule AFM

通过单分子 AFM 进行纳米级 DNA 诊断

• 批准号: 6944800
• 负责人: PIOTR E MARSZALEK
• 金额: $ 18.48万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6944800
• 关键词: DNA; DNA damage; DNA repair; Escherichia coli; atomic force microscopy; bacterial genetics; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biomedical equipment development; elasticity; human genetic material tag; method development; nanotechnology; nucleic acid chemical synthesis; radiobiology; technology /technique development; ultraviolet radiation

DESCRIPTION (provided by applicant): DNA damage is the first step in the initiation of cancer. Every human cell experiences approximately 50,000 base damage/removal events and ~1000-2000 base mispair errors per day, Since DNA alterations vary from one DNA molecule to another it is important to detect such lesions in individual molecules. The primary objective of this R21 proposal is to develop a methodology for detecting DNA lesions at the single molecule level using Atomic Force Microscopy (AFM) techniques. Lesions will be detected either directly, by force spectroscopy and/or by imaging with carbon nanotube (SWNT) AFM tips, or indirectly, by detecting the secondary changes induced in DNA by the repair proteins. These secondary changes include e.g. strand incision, unwinding, degradation and re-synthesis. They are co-localized with the primary lesion and should be easily detected by our AFM assay. To achieve our objective we need to optimize the AFM platform to achieve: Angstrom level precision in the x, y, and z-axis and force errors below 10 pN. The platform must be able to align a molecule with the pulling direction and an ability to set a limit on the pulling force in order to preserve the coupling of the molecule to the instrument. We will construct various DNA molecules with model lesions and use AFM imaging and force spectroscopy to determine their fingerprints. We will study the effect of UV radiation on DNA elasticity and the course of its photoreactivation. A second objective is to explore the use of AFM spectroscopy and imaging to study the mismatch repair (MMR) in E. coil and in humans. We will determine force spectrograms of DNA molecules containing a mismatched base in the presence of bacterial and human repair activities. We will record the changes of DNA elasticity during the course of repair. We will also visualize primary lesions, repair-induced secondary alterations and DNA/proteins complexes. These measurements will allow us to examine key intermediates in the repair reaction and to further clarify molecular functions of the repair proteins and their assemblies. The proposed experiments will use for the first time, AFM-based single-molecule force spectroscopy to detect DNA lesions and to examine the course of a repair reaction. The findings should be of great significance for nanoscale DNA diagnostics and DNA damage and repair research.

描述（由申请人提供）：DNA损伤是癌症开始的第一步。每个人类细胞每天经历约50,000个基本损伤/去除事件和〜1000-2000的基本错误误差，因为DNA的改变从一个DNA分子到另一个DNA分子不等，因此在单个分子中检测此类病变很重要。该R21建议的主要目的是开发一种使用原子力显微镜（AFM）技术在单分子水平检测DNA病变的方法。通过力光谱和/或通过碳纳米管（SWNT）AFM TIPS或间接检测通过修复蛋白在DNA诱导的二次变化来直接检测病变。这些次要变化包括例如链切口，放松，退化和重新合成。它们与原发性病变共定位，应由我们的AFM分析轻松检测。为了实现我们的目标，我们需要优化AFM平台以实现：X，Y和Z轴中的Angstrom级别的精度以及低于10 pn的力误差。该平台必须能够将分子与拉力方向对齐，并能够对拉力限制，以保持分子与仪器的耦合。我们将使用模型病变构建各种DNA分子，并使用AFM成像和力光谱来确定其指纹。我们将研究紫外线辐射对DNA弹性及其光反应过程的影响。第二个目标是探索AFM光谱和成像研究在E.卷轴和人类中研究不匹配修复（MMR）的使用。我们将确定在存在细菌和人类修复活性的情况下含有不匹配碱的DNA分子的力谱图。我们将记录维修过程中DNA弹性的变化。我们还将可视化原发性病变，修复诱导的次要改变和DNA/蛋白质复合物。这些测量值将使我们能够检查修复反应中的关键中间体，并进一步阐明修复蛋白及其组件的分子功能。提出的实验将首次使用基于AFM的单分子力光谱来检测DNA病变并检查修复反应的过程。这些发现对于纳米级DNA诊断和DNA损伤和修复研究应该具有重要意义。