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

并不是所有的DNA都有熟悉的双螺旋形状。DNA的某些区域富含四种构成成分之一的鸟嘌呤，形成一种不同的四股结构，称为G-四链(G4)。在人类基因组中，有70多万个G4形成区，其中约10,000个在细胞中形成G4S。一些G4S干扰DNA复制、DNA修复和RNA转录。该项目将结合最先进的DNA测序技术和荧光显微镜，将人类基因组中的所有G4区域连接到固体表面，并研究它们与其他分子的相互作用。该项目将为爱荷华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-四联体在调节DNA复制、DNA修复和RNA转录方面发挥着重要作用。该项目将提供一个技术平台，用于并行分析和比较可能在富含G的序列中形成的数千种潜在结构。人的G4染色质将使用特定的抗体进行免疫沉淀，并使用Illumina MiSeq下一代测序流式细胞进行测序。它将以一种非常规的方式使用，而不是在测序后处理流动细胞：在测序过程中克隆扩增的单链DNA将被允许重新折叠成G4结构，并将使用改进的TIRF显微镜来可视化和量化折叠的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，将重复显示G4。为了增加STEM研究的多样性和兴奋度，该团队将继续参与机构外展计划，为本科生和高中生以及来自爱荷华州不提供博士课程的本科院校的教职员工和学生提供研究机会。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。

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.