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

University Of Iowa, Iowa City IA

Investigators

Abstract

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.