Nanopore Detection of DNA and RNA Modifications

 描述(由申请人提供)：DNA和RNA碱基的化学修饰是对内源性和环境暴露于活性物种(如氧化剂和烷化剂)的反应，以及在安装表观遗传标记期间发生的。虽然关于表观遗传碱基改变的类型和位置的大量信息可以从亚硫酸氢盐测序中收集到，但这种方法并没有被常规地用于氧化应激修饰。这个项目调查了离子通道蛋白的使用，包括野生型和工程化的，作为纳米孔平台的一部分，以检测DNA：DNA双链或DNA：RNA双链中碱基修饰的存在。这一假说建立在这些实验室的最新结果之上，这些结果表明，当双链DNA被电泳性地驱动到蛋白质腔的前厅时，阿尔法溶血素离子通道的“闩锁区”是碱基对变化的灵敏检测器。这项工作提出，结合蛋白质的定点突变和化学修饰，再加上对蛋白质-核酸-电解质相互作用的生物物理理解，可以微调离子通道的反应，以感知核酸双链的变化。其具体目的是(1)优化α-溶血素的闩锁区域，以检测DNA：RNA双链中的碱基修饰；(2)构建DNA探针并检测tRNAs反密码子区域的氧化损伤；(3)探索γ-溶血素作为检测双链DNA移位中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切割和翻译抑制的途径。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，报告作为饮食、药物、炎症和疾病状态功能的DNA或RNA碱基修饰的技术在现代医学中至关重要。该项目的创新方面包括一种以保留损伤部位信息的方式对DNA损伤进行聚合酶链式反应扩增的新方法，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。

 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.