DNA Photoproducts as Intrinsic Probes of Non-B DNA Conformations

DNA 光产物作为非 B DNA 构象的内在探针

• 批准号: 2003688
• 负责人: John-Stephen Taylor
• 金额: $ 45万
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2003688&HistoricalAwards=false
• 关键词: DNA; Photoproducts; Intrinsic; Probes; Non

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. John-Stephen Taylor at Washington University-Saint Louis to develop methods for establishing the existence and locations of G-quadruplexes in cells. The double-helical B-DNA structure discovered by Watson and Crick revolutionized the understanding of how genetic information is replicated and expressed as RNA and proteins in cells. G-quadruplexes are labile four-stranded DNA structures that have more recently been found, along with other non-B-DNA structures, to add new dimensions to the fundamental understanding of how the expression of genetic information is controlled. The study uses a unique reaction of G-quadruplexes with light that leads to indelible and unique molecular imprints in DNA. These distinctive imprints provide direct evidence for the existence of G-quadruplexes in cells, allowing their positions and dynamic behavior to be investigated within the human genome. The existence of G-quadruplexes and other non-B DNA structures lead to better understanding of their roles in disease development. Graduate students working on this project receive multidisciplinary training in synthetic organic chemistry, photochemistry, automated DNA synthesis, molecular and cellular biology, and mass spectrometry. In addition, teaching modules and tutorials for high school students are developed that use physical and computer models to understand G-quadruplex structures and how their photochemistry differs from that of standard B-DNA. This project builds on a long-standing program in the principal investigator's laboratory to use chemical and biophysical tools to study the details of complex nucleic acis structures, with a particular focus on G-quadruplex structures. The scientific approach takes advantage of the observation that UV irradiation of G-quadruplex-forming sequences containing thymine (T) and cytosine (C) bases in adjacent lateral loops results in the formation of stable anti-cyclobutane pyrimidine dimers (anti-CPDs), photoproducts that cannot form in canonical B-DNA. Highly sensitive methods are developed to detect these anti-CPDs photoproducts in cells using post-labeling and isotope dilution mass spectrometric methods. Their positions within the genome are located through ligation-mediated polymerase chain reaction (PCR) methods. Structure-photoreactivity relationships that serve as unique molecular signatures of G-quadruplexes and other related non-B DNA conformations are determined using enzyme-coupled mass spectrometry assays of site-specific isotopically labeled DNA. The broader impacts include presenting research opportunities to students from traditionally underrepresented communities and the development of new materials to teach concepts of DNA structure that align with educational standards.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

有了这个奖项，化学部门的生命过程化学项目资助了华盛顿大学圣路易斯分校的约翰-斯蒂芬·泰勒博士，以开发确定细胞中g -四重体存在和位置的方法。沃森和克里克发现的双螺旋B-DNA结构彻底改变了人们对遗传信息如何在细胞中以RNA和蛋白质的形式复制和表达的理解。g -四联体是最近发现的不稳定的四链DNA结构，与其他非b -DNA结构一起，为遗传信息的表达如何被控制的基本理解增加了新的维度。这项研究利用一种独特的g -四联体与光的反应，在DNA中产生不可磨灭的独特分子印记。这些独特的印记为细胞中g -四重体的存在提供了直接证据，使它们的位置和动态行为可以在人类基因组中进行研究。g -四联体和其他非b DNA结构的存在有助于更好地理解它们在疾病发展中的作用。从事该项目的研究生接受合成有机化学、光化学、自动DNA合成、分子和细胞生物学以及质谱学等多学科培训。此外，为高中学生开发的教学模块和教程使用物理和计算机模型来理解g -四重体结构及其光化学与标准B-DNA的不同之处。该项目建立在首席研究员实验室的一个长期计划的基础上，该计划使用化学和生物物理工具来研究复杂核酸结构的细节，特别关注g-四重结构。该科学方法利用了以下观察结果：紫外线照射相邻侧环中含有胸腺嘧啶(T)和胞嘧啶(C)碱基的g -四聚体形成序列，可形成稳定的抗环丁烷嘧啶二聚体（anti-CPDs），这种光产物不能在标准B-DNA中形成。利用后标记和同位素稀释质谱方法，开发了高灵敏度的方法来检测细胞中这些抗cpds光产物。它们在基因组中的位置是通过连接介导的聚合酶链反应（PCR）方法定位的。结构-光反应性关系作为g -四联体和其他相关非b DNA构象的独特分子特征，使用酶偶联质谱法测定位点特异性同位素标记DNA。更广泛的影响包括为来自传统上代表性不足的社区的学生提供研究机会，以及开发符合教育标准的新材料来教授DNA结构概念。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。