Instrument for Single Molecule Sequencing of Alzheimer's-Relevant Genomic Oxidati

用于阿尔茨海默病相关基因组氧化的单分子测序仪器

• 批准号: 8464401
• 负责人: David E. Wolf
• 金额: $ 30万
• 依托单位: RADIATION MONITORING DEVICES, INC.
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8464401
• 关键词: Affect; Age; Alzheimer' s Disease; Area; Base Pairing; Base Sequence; Cell physiology; Cells; Clinical; DNA; DNA Damage; DNA Modification Process; DNA Sequence; Deamination; Detection; Development; Diagnosis; Diagnostic; Discrimination; Disease; Equipment; Etiology; Evaluation; Future; Genes; Genome; Genomics; Government; Heart Diseases; Hippocampus (Brain); In Vitro; Influentials; Label; Lasers; Length; Lesion; Longevity; Malignant Neoplasms; Mitochondria; Modification; Molecular; Nanotechnology; Neurofibrillary Tangles; Neurologic; Neurons; Nuclear; Nucleic Acids; Optics; Oxidative Stress; Parkinson Disease; Pathogenesis; Patients; Persons; Phase; Play; Ploidies; Process; Prophylactic treatment; Protocols documentation; Radiation Monitoring; Raman Spectrum Analysis; Reading; Research; Resolution; Role; Sampling; Scanning; Scientist; System; Techniques; Technology; Texas; Tissues; Traumatic Brain Injury; United States National Institutes of Health; Universities; Work; base; design; drug discovery; ds-DNA; hippocampal pyramidal neuron; instrument; interest; laser capture microdissection; monitoring device; new technology; nucleobase; oxidation; oxidative damage; promoter; research study; senescence; single molecule; tool

DESCRIPTION (provided by applicant): DNA damage threatens the longevity and functionality of cells, and is common to the pathogenesis of many diseases. Better tools are needed for clinical identification and research into mechanisms underlying DNA oxidation, deamination, and hyper/hypomethylation, and the role these lesions play in the genesis and etiology of Alzheimer's disease. All existing and proposed techniques of DNA sequencing are insensitive to a broad spectrum of disease-relevant types of nucleobase damage Oxidative stress is one of the most earliest and prominent features of Alzheimer disease. Prior work of our collaborator and consultant, Dr. George Perry at University of Texas San Antonio and Dr. Xiongwei Zhu at Case Western Reserve University, demonstrated increased oxidative damage to nucleic acid in vulnerable neurons in the hippocampal tissues from Alzheimer patients. Due to the limits of technology available, it has not been possible to conclusively demonstrate oxidation in DNA is an upstream cause of AD and neuronal damage. It is also critical that vulnerable and influential genes, promoter, and sequences be identified to guide clinical prophylaxis and treatment. RMD Inc. is working to develop a label-free single-molecule-sensitive optical sequencing technology, capable of locating modified bases within single or double stranded DNA molecules in vitro. This technology will be capable of identifying and localizing genomic damage, while taking advantage of long read lengths and direct sequencing of unamplified DNA. In particular, this technology will use Raman spectroscopy with AFM (Tip-Enhanced Raman Spectroscopy) to directly read immobilized DNA for oxidized or otherwise non-canonical bases (i.e. methylated de-aminated, alkylated, etc.). The development of the new technology as proposed is a much needed one to further oxidative stress study in AD research. Our Phase I exploratory research will prove the principle of damage detection, and determine the initial resolution of the sequencing technique. Optics will be optimized, and the equipment will be evaluated for the capability to achieve single-base resolution in Phase II. In Phase II, RMD will achieve single base resolution, and apply the technology to the analysis of AD-derived DNA. The lab of Dr. Xiongwei Zhu will isolate pyramidal neurons in the CA1 area of hippocampus from well characterized AD patients and age-matched control patients by laser captured microdissection and extract DNA from these neurons and provide the DNA to RMD Inc. for further characterization of DNA oxidation. Experiments will be planned and directed by both Dr. George Perry and scientists at RMD. PUBLIC HEALTH RELEVANCE: Radiation Monitoring Devices, Inc., proposes to develop a single molecule DNA sequencing nanotechnology sensitive to a wide variety of DNA modifications, including those central to the pathogenesis of Alzheimer's, Parkinson's and heart diseases, as well as traumatic brain injury. The technology will be used clarify the relationship between sequence specific oxidative damage and Alzheimer's disease, and as a result is a revolutionary tool to direct future therapies and diagnostics. This technology provides unique capabilities as a research tool for understanding gene control of cell function, for drug discovery and ultimately for diagnosis of a broad range of neurologic diseases, senescence and cancer.

描述（由申请人提供）：DNA 损伤威胁细胞的寿命和功能，并且是许多疾病的发病机制中常见的。需要更好的工具来临床鉴定和研究 DNA 氧化、脱氨基和高甲基化/低甲基化的机制，以及这些病变在阿尔茨海默病的发生和病因学中所起的作用。所有现有和拟议的 DNA 测序技术对多种与疾病相关的核碱基损伤类型都不敏感氧化应激是阿尔茨海默病最早和最突出的特征之一。我们的合作者和顾问，德克萨斯大学圣安东尼奥分校的 George Perry 博士和凯斯西储大学的 Xiongwei Zhu 博士之前的研究表明，阿尔茨海默病患者海马组织中脆弱神经元的核酸氧化损伤增加。由于现有技术的限制，尚无法最终证明 DNA 氧化是 AD 和神经元损伤的上游原因。同样重要的是，鉴定脆弱和有影响的基因、启动子和序列以指导临床预防和治疗。 RMD Inc. 正在致力于开发一种无标记单分子敏感光学测序技术，能够在体外定位单链或双链 DNA 分子内的修饰碱基。该技术将能够识别和定位基因组损伤，同时利用长读长和未扩增 DNA 的直接测序。特别是，该技术将使用拉曼光谱与 AFM（尖端增强拉曼光谱）直接读取固定 DNA 中的氧化碱基或其他非规范碱基（即甲基化脱氨基、烷基化等）。所提出的新技术的发展对于进一步研究 AD 研究中的氧化应激是非常必要的。 我们的第一阶段探索性研究将证明损伤检测的原理，并确定测序技术的初始分辨率。第二阶段将优化光学器件，并评估设备实现单碱基分辨率的能力。 在第二阶段，RMD将实现单碱基分辨率，并将该技术应用于AD衍生DNA的分析。朱雄伟博士的实验室将通过激光捕获显微切割技术，从特征明确的 AD 患者和年龄匹配的对照患者中分离出海马 CA1 区的锥体神经元，并从这些神经元中提取 DNA，并将 DNA 提供给 RMD Inc. 以进一步表征 DNA 氧化。实验将由 George Perry 博士和 RMD 的科学家共同计划和指导。 公共健康相关性：辐射监测设备公司提议开发一种对多种 DNA 修饰敏感的单分子 DNA 测序纳米技术，其中包括对阿尔茨海默病、帕金森病、心脏病以及创伤性脑损伤发病机制至关重要的修饰。该技术将用于阐明序列特异性氧化损伤与阿尔茨海默病之间的关系，因此是指导未来治疗和诊断的革命性工具。 该技术作为研究工具提供了独特的功能，可用于了解细胞功能的基因控制、药物发现以及最终诊断各种神经系统疾病、衰老和癌症。