DNA Photoproducts as Intrinsic Probes of Non-B DNA Conformations

该奖项将资助华盛顿大学圣路易斯分校的John-Stephen Taylor博士开发确定细胞中G-四链体的存在和位置的方法。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复制和表达为RNA和蛋白质的理解。G-四链体是最近发现的不稳定的四链DNA结构，与其他非B-DNA结构一起被发现，以增加对遗传信息表达如何控制的基本理解的新维度。这项研究使用了G-四链与光的独特反应，从而在DNA中形成了不可磨灭的独特分子印记。这些独特的印记为细胞中G-四链体的存在提供了直接证据，使人们能够在人类基因组中研究它们的位置和动态行为。G-四链体和其他非B-DNA结构的存在使人们更好地了解它们在疾病发展中的作用。从事这个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方面的多学科培训。此外，还为高中生开发了教学模块和教程，使用物理和计算机模型来理解G-四链结构，以及它们的光化学与标准B-DNA的不同之处。这个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的核ACIs结构的细节，特别是G-四链结构。这种科学方法利用了这样的观察结果，即在相邻侧环中含有胸腺嘧啶(T)和胞嘧啶(C)碱基的G-四链形成序列在紫外线照射下会形成稳定的抗环丁烷嘧啶二聚体(anti-CPDS)，这些光产物不能在标准的B-DNA中形成。利用后标记和同位素稀释质谱学方法，开发了高灵敏度的方法来检测细胞中的这些抗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式反应(PCR)方法定位的。作为G-四链体和其他相关非B DNA构象的唯一分子特征的结构-光反应性关系是使用位点特异的同位素标记DNA的酶耦合质谱分析来确定的。更广泛的影响包括向传统上代表性不足的社区的学生提供研究机会，以及开发新材料来教授与教育标准一致的DNA结构概念。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.