In situ detection of stalled cleavage complexes for studies in aging

用于衰老研究的停滞裂解复合体的原位检测 摘要 该项目将介绍并验证第一种检测停滞共价裂解的定量原位方法 在哺乳动物细胞正常老化过程中自发产生的复合体(SCCs)。这些 共价拓扑异构酶-DNA链断裂中间产物不断产生并缓慢积累在 老化的细胞。与年龄相关的退行性疾病，如阿尔茨海默氏症，会加速它们的形成。 SCCs是在流产的催化作用下产生的，最近被发现是衰老过程的关键部分 在真核生物中。尽管干细胞在正常衰老过程和病理过程中起着重要的作用 与衰老有关，目前还没有检测和定量这些DNA的具体方法 原位改变与个体复合体的敏感性。现有的大宗生化方法不是 适用于原位格式，如固定细胞和组织切片。 在这个项目中，我们将通过开发一种新的使能分子技术来克服这一限制 对老龄化的分析。创新的原位方法将基于新的标记原理，并将 同时联合检测SCC的几个特征特征。 这项研究将缩小一项重大的技术差距，并将推出第一个定量检测方法 原位鳞状细胞的评估。该项目将为衰老和衰老相关的分子研究创造一个新的工具 疾病，在生物医学中具有广泛和普遍的用途。 该项目将达到以下具体目标： 1.介绍第一种检测共价停滞裂解复合体(SCCs)的原位定量方法， 它们在哺乳动物细胞正常老化的过程中自发产生。测试和验证标签的步骤 新技术的原理，通过使用现场模型和SCCs的规范生产。为了确保 新方法的定量能力及其对SCC的高度特异性。 2.在细胞和组织切片模型中对新方法进行生化和活体验证 SCC的生成。评估和确认新方法的量化能力及其高度特异性 适用于SCC。以确保在各种样品中的稳健和可靠的分析性能。

In situ detection of stalled cleavage complexes for studies in aging Abstract This project will introduce and validate the first quantitative in situ approach detecting stalled covalent cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. These covalent topoisomerase-DNA strand break intermediates are continually produced and slowly accumulate in aging cells. Their formation is accelerated by age-related degenerative diseases, such as Alzheimer’s. SCCs are generated in abortive catalysis and were recently revealed to be a critical part of the aging process in eukaryotes. In spite of the essential role of SCCs in the normal aging process and in the pathologies associated with aging, at present time, there are no specific approaches detecting and quantitating these DNA alterations in situ with the individual complex sensitivity. The existing bulk biochemical approaches are not applicable to the in situ formats, such as fixed cells and tissue sections. In this project, we will overcome this limitation by developing a new and enabling technology for molecular analysis of aging. The innovative in situ method will be based on novel labeling principles, and will simultaneously co-detect several characteristic features of SCCs. This study will close a significant technological gap and will introduce the first assay for quantitative assessment of SCCs in situ. The project will create a new tool for molecular research in aging and age-related disorders, with broad and general utility in biomedicine at large. The project will reach these Specific Aims: 1. To introduce the first quantitative in situ approach detecting covalent stalled cleavage complexes (SCCs), which spontaneously originate in mammalian cells during their normal aging. To test and validate the labeling principles of the new technology by using in situ models with regulated production of SCCs. To ensure the quantitative ability of the new approach and its high specificity for SCCs. 2. To test and validate the new method in the cell and tissue section models with biochemical and in vivo generation of SCCs. To assess and confirm the quantitative ability of the new approach and its high specificity for SCCs. To ensure robust and reliable assay performance in various samples.