EAGER: G4-TIRFM, a platform for massively parallel analysis of non-canonical DNA structures

并不是所有的DNA都具有我们所熟悉的双螺旋结构。DNA的某些区域富含四种结构单元之一的鸟嘌呤，形成一种不同的四链结构，称为G-四链体（G4）。在人类基因组中，有超过700，000个G4形成区域，其中约10，000个被证明在细胞中形成G4。一些G4干扰DNA复制、DNA修复和RNA转录。本计画将联合收割机结合最先进的DNA定序技术与萤光显微镜技术，将人类基因组中所有的G4区域连接到固体表面上，并研究它们与其他分子的相互作用。该项目将为爱荷华州大学通过几个外展机制招募的本科生和高中生提供研究经验。G-四链体在调节DNA复制、DNA修复和RNA转录方面发挥重要作用。该项目将提供一个技术平台，用于对富G序列中可能形成的数千种潜在结构进行并行分析和比较。将使用特异性抗体对人G4染色质进行免疫沉淀，并使用Illumina MiSeq下一代测序流动池进行测序。在测序后不处理流动池，而是以非常规方式使用：在测序过程中克隆扩增的ssDNA将被允许重新折叠成G4结构，并且改良的TIRF显微镜将用于可视化和定量折叠的G4 DNA簇。在用DNA聚合酶、聚合酶/解旋酶组合和其他G4结合剂处理后，将重复G4的可视化。为了增加STEM研究的多样性和兴奋性，该团队将继续参与机构外展计划，为本科生和高中生提供研究机会，以及来自不提供博士课程的爱荷华州本科院校的教师和学生。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值进行评估来支持和更广泛的影响审查标准。

Not all DNA has the familiar double-helix shape. Some regions of DNA rich in one of the four building blocks, guanine, form a different, four stranded kind of structure, called G-quadruplex (G4). Th the human genome, there are more than 700,000 G4-forming regions, and about 10,000 of them were shown to form G4s in cells. Some G4s interfere with DNA replication, DNA repair and RNA transcription. This project will combine the state of the art DNA sequencing technology with fluorescent microscopy to attach all G4 regions in human genome to the solid sorface and study their interactions with other molecules. The project will provide research experience for the undergraduates and high school students recruited through several outreach mechanisms at the University of Iowa.G-quadruplexes play important roles in regulating DNA replication, DNA repair and RNA transcription. This project will provide a technology platform for the parallel analysis and comparison of thousands of potential structures that can form within the G-rich sequences. Human G4 chromatin will be immunoprecipitated using specific antibodies and sequenced using an Illumina MiSeq next generation sequencing flow cells. Instead of disposing the flow cell after sequencing, it will be employed in a non-conventional way: ssDNA that has been clonally amplifed in the sequencing process will be allowed to refold into G4 structures, and modified TIRF microscope will be used to visualize and quantify clusters of folded G4 DNA. Visualization of G4 will be repeated after treatments with DNA polymerases, polymerase/helicase combinations and other G4 binders. To increase the diversity in and excitement about STEM research, the team will continue their involvement with the institutional outreach programs to provide research opportunities to undergraduate and high school students, as well as faculty and students from Iowa undergraduate institutions that do not offer doctoral programs.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.