Nanopore Detection of DNA and RNA Modifications

DNA 和 RNA 修饰的纳米孔检测

• 批准号: 8887872
• 负责人: Cynthia J Burrows
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8887872
• 关键词: Alkylating Agents; Bar Codes; Base Pairing; Base-Base Mismatch; Cations; Cells; Cellular Stress; Chemicals; Codon Nucleotides; Cysteine; Detection; Diet; Discrimination; Disease; Electrolytes; Engineering; Environmental Exposure; Epigenetic Process; Escherichia coli; Exposure to; Glean; Goals; Guanine; Hemolysin; Inflammation; Ion Channel; Ion Channel Protein; Laboratories; Lead; Length; Lesion; Location; Maps; Measurement; Membrane; Methodology; Methods; Micronutrients; Mitochondria; Modern Medicine; Modification; Monitor; Mutagenesis; Nucleic Acids; Oxidants; Oxidative Stress; Pathway interactions; Pattern; Pharmaceutical Preparations; Post-Translational Protein Processing; Proline-Specific tRNA; Proteins; Protocols documentation; RNA; Reading; Reporting; Research; Resolution; Rest; Ribonucleases; Site; Site-Directed Mutagenesis; Staging; Tail; Technology; Temperature; Testing; Toxic Environmental Substances; Transfer RNA; Translations; Vestibule; Work; antioxidant therapy; base; bisulfite sequencing; constriction; design; detector; ds-DNA; epigenetic marker; innovation; insight; nanopore; novel; oxidation; oxidative damage; public health relevance; research study; response; screening; single molecule; tool; voltage

 DESCRIPTION (provided by applicant): Chemical modifications to DNA and RNA bases occur in response to endogenous and environmental exposure to reactive species such as oxidizing and alkylating agents as well as during installation of epigenetic markers. Although considerable information about the type and location of epigenetic base changes can be gleaned from bisulfite sequencing, no such methodology is routinely employed for oxidative stress modifications. This project investigates the use of ion channel proteins, both wild-type and engineered, as part of a nanopore platform to detect the presence of base modifications in DNA:DNA duplexes or DNA:RNA duplexes. The hypothesis rests on recent results from these laboratories showing that the "latch zone" of the alpha-hemolysin ion channel is a sensitive detector of changes in base pairs when double- stranded DNA is electrophoretically driven into the vestibule of the protein cavity. The work proposes that a combination of site-directed mutagenesis and chemical modification of the protein, combined with a biophysical understanding of the protein-nucleic acid-electrolyte interactions, can fine-tune the response of the ion channel for sensing changes in nucleic acid duplexes. The specific aims are to (1) optimize the latch zone of alpha-hemolysin to sense base modifications in DNA: RNA duplexes, (2) construct DNA probes and examine oxidative damage in the anti-codon region of tRNAs, and (3) explore gamma-hemolysin as a tool to examine DNA damage in translocating double-stranded DNA. A key aspect of the work is to provide a new single-molecule method to examine changes in the bases of transfer RNA that will provide insight into the pathway by which oxidative stress results in tRNA cleavage and inhibition of translation. Given the significant correlations between oxidative stress and disease, and the public focus on micronutrients and antioxidant therapy, technologies that report on modifications to DNA or RNA bases as a function of diet, drugs, and inflammation and disease state are of key importance in modern medicine. Innovative aspects of the project include a novel method for PCR amplification of DNA damage in a way that retains information about the sites of damage, and the use of non-traditional components of hemolysin-type ion channels for sensing of DNA:DNA and DNA:RNA duplexes.

 描述（由申请人提供）：DNA和RNA碱基的化学修饰发生在内源性和环境暴露于反应性物质（如氧化剂和烷化剂）以及表观遗传标记安装期间。虽然可以从亚硫酸氢盐测序中收集到关于表观遗传碱基变化的类型和位置的大量信息，但没有这种方法常规用于氧化应激修饰。该项目研究使用离子通道蛋白，野生型和工程化的，作为纳米孔平台的一部分，以检测DNA：DNA双链体或DNA：RNA双链体中碱基修饰的存在。该假设基于来自这些实验室的最近结果，该结果显示α-溶血素离子通道的“闩锁区”是当双链DNA被电泳驱动到蛋白质腔的前庭中时碱基对变化的灵敏检测器。这项工作提出，结合定点诱变和化学修饰的蛋白质，结合生物物理学的理解的蛋白质-核酸-电解质相互作用，可以微调的离子通道的响应，用于传感核酸双链体的变化。具体目标是（1）优化α-溶血素的锁区以检测DNA：RNA双链体中的碱基修饰，（2）构建DNA探针并检测tRNA反密码子区域的氧化损伤，以及（3）探索γ-溶血素作为检测易位双链DNA中DNA损伤的工具。这项工作的一个关键方面是提供一种新的单分子方法来检查转移RNA碱基的变化，这将有助于深入了解氧化应激导致tRNA切割和抑制翻译的途径。鉴于氧化应激与疾病之间的显著相关性，以及公众对微量营养素和抗氧化治疗的关注，报告DNA或RNA碱基修饰作为饮食，药物，炎症和疾病状态的功能的技术在现代医学中具有关键重要性。该项目的创新方面包括一种新的DNA损伤PCR扩增方法，该方法保留了有关损伤部位的信息，以及使用溶血素型离子通道的非传统成分来检测DNA：DNA和DNA：RNA双链体。