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Nanoscale DNA Diagnostics by Single Molecule AFM

通过单分子 AFM 进行纳米级 DNA 诊断

基本信息

批准号：
7118808
负责人：
PIOTR E MARSZALEK
金额：
$ 18.05万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-09-01 至 2008-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7118808
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

项目摘要

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损伤是癌症发生的第一步。每个人类细胞每天经历大约50,000个碱基损伤/移除事件和约1000-2000个碱基错配错误。由于DNA改变在不同的DNA分子之间是不同的，因此检测单个分子中的这种损伤是很重要的。本R21提案的主要目标是开发一种使用原子力显微镜（AFM）技术在单分子水平上检测DNA病变的方法。损伤可以通过力谱和/或碳纳米管（SWNT） AFM尖端成像直接检测，也可以通过检测修复蛋白在DNA中诱导的二次变化间接检测。这些次生变化包括：链切割、解绕、降解和再合成。它们与原发性病变共定位，应该很容易被我们的AFM检测到。为了实现我们的目标，我们需要优化AFM平台，以实现：在x， y和z轴上的埃级精度和低于10 pN的力误差。该平台必须能够将分子与拉力方向对齐，并能够设置拉力限制，以保持分子与仪器的耦合。我们将构建具有模型病变的各种DNA分子，并使用AFM成像和力谱来确定其指纹图谱。我们将研究紫外线辐射对DNA弹性的影响及其光活化过程。第二个目标是探索使用AFM光谱和成像来研究e线圈和人类的错配修复（MMR）。我们将确定在细菌和人类修复活动中含有不匹配碱基的DNA分子的力谱图。我们将记录修复过程中DNA弹性的变化。我们还将可视化原发性病变，修复诱导的继发性改变和DNA/蛋白质复合物。这些测量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装的分子功能。拟议的实验将首次使用基于原子力显微镜的单分子力光谱来检测DNA损伤并检查修复反应的过程。这一发现对纳米尺度DNA诊断和DNA损伤与修复研究具有重要意义。