EpiSeq · Single molecule sequencing and biophysical properties of oxidized genomic DNA using magnetic tweezers.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2019-07-01 → 2021-06-30
- EU contribution
- €184,708
- Participants
- 1
- Scheme
- MSCA-IF-EF-RI
Lines connect the coordinator with its partners.
Results in brief
Single molecule sequencing and biophysical properties of oxidized genomic DNA using magnetic tweezers.
Oxidative stress is a persistent threat to genomic DNA and RNA and is associated with major causes of human mortality, including cancers, diabetes and aging. 8-oxoguanine (8-oxoG) is the most common product of oxidative stress in nucleic acids. Evidence has accumulated to show that 8-oxoG is not only a pre-mutagenic DNA base lesion but also has essential roles in the regulation of gene expression and may act as an epigenetic mark. For such functions, the oxidative modification would need to occur in regulatory regions of genome such as promoters. Indeed, in vitro and genome-wide studies of the distribution of 8-oxoG showed that potential G4-forming sequences (repetitive guanine-rich sequences which can form G-quadruplex (G4) secondary structures), presented in ~50% promoters of human genes, are preferential substrates for oxidation. However, the mechanism of how 8-oxoG regulates gene transcription remains unclear. In addition, some recent studies showed contradicting conclusions concerning the distribution of 8-oxoG at gene regulatory regions, which makes the role of 8-oxoG in gene regulation an intense area of research. The main objectives of this project are: 1) study in vitro the effect of 8-oxoG modification on G-quadruplex (G4) folding at biochemical and biophysical level 2) develop a reliable method to capture a specific genomic fragment, starting from yeast genomic DNA 3) develop the single-molecule technology to detect the presence and location of oxidize guanines in the DNA molecules, at single-nucleotide resolution
Data: CORDIS, © European Union
Project objective
Next-generation DNA sequencing technologies are at the core of modern molecular biology and are rapidly entering the technological arsenal for personalized medicine. The recent explosion of sequencing technologies has provided researchers with various solutions to fit their sequencing needs, with a large spectrum of costs and limitations. However, most sequencing methods use enzymatic duplication of DNA to generate a strong enough sequencing signal. Therefore, chemical modifications potentially present on native bases are lost, and with them, an entire level of epigenetic information about. Although some workarounds are emerging, they are indirect, costly, and not yet amenable for the targeted sequencing of a given native genomic locus or biophysical analysis of the modified DNA. Given the need to understand the epigenetic layer of genetic information, I propose to develop a reliable and cheap method to map, at the single-molecule resolution, the epigenetic status of a genomic DNA locus of interest. This will be achieved through biochemical capture of this locus using CRISPR/Cas9 technology, and its epigenetic sequencing via single-molecule manipulation by magnetic tweezers. In addition to identifying the nature and the position of the modified bases at a given genomic DNA locus, I will use this magnetic tweezer technology to address fundamental questions about the effect of epigenetic modifications on the biophysical properties of DNA (formation of DNA structures and protein interactions). Specifically, I will focus on 8-oxoguanine, for which there is increasing interest since it is a hallmark of degenerative pathologies like cancer or Alzheimer’s Disease, and since it has recently entered the known epigenetic arsenal. This project has great potential applications in medical diagnosis. It will give me a first-hand experience in developing a cutting-edge technology, which will be of great benefit to my future independent research career.
Original text from CORDIS.
Participants
- MUSEUM NATIONAL D'HISTOIRE NATURELLE · ParisCoordinatorFrance
Links
Data: CORDIS, © European Union