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

描述（由申请人提供）：DNA损伤威胁到细胞的寿命和功能，是许多疾病的常见发病机制。临床鉴定和研究DNA氧化、脱氨和高/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，需要更好的工具。所有现有的和提出的DNA测序技术对广泛的疾病相关的核碱基损伤类型不敏感。氧化应激是阿尔茨海默病最早期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博士之前的工作表明，阿尔茨海默病患者海马组织中易损神经元的核酸氧化损伤增加。由于现有技术的限制，还不可能最终证明DNA氧化是阿尔茨海默病和神经元损伤的上游原因。鉴定易受伤害和有影响的基因、启动子和序列以指导临床预防和治疗也是至关重要的。RMD公司正在开发一种无标记的单分子敏感光学测序技术，能够在体外定位单链或双链DNA分子中的修饰碱基。这项技术将能够识别和定位基因组损伤，同时利用长读取长度和未扩增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的氧化或其他非规范碱基（即甲基化去胺化，烷基化等）。本文所提出的新技术的开发是进一步开展AD氧化应激研究的必要条件。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步解决方案。光学将被优化，设备将被评估在第二阶段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解剖从特征明确的AD患者和年龄匹配的对照患者的海马CA1区分离锥体神经元，并从这些神经元中提取DNA，并将DNA提供给RMD公司进一步表征DNA氧化。实验将由乔治·佩里博士和麻省理工学院的科学家共同规划和指导。

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.