N6MeA ChemSeq · Development of chemical methods for DNA N6-methyladenine mapping
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2018-04-19 → 2020-04-18
- EU contribution
- €183,455
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Development of chemical methods for DNA N6-methyladenine mapping
Core genetic information is stored as a specific sequence of the DNA alphabet. The four canonical bases constituting the letters of the DNA alphabet can undergo small changes in their chemical structures to add an additional layer of information on the genome. These modifications allow organisms to use their genome in different ways without altering the core genetic sequence. This is central for living systems to adapt to environmental changes, but also for development and differentiation to different cellular identities. For a better understanding of the development and functioning of organisms and the occurrence of disease, understanding the biological roles of these DNA modifications is fundamental. One of these modified DNA bases is N6-methyladenine (N6MeA), which is present at high levels in bacterial genomes and at considerably lower levels in the DNA of eukaryotes. Recent studies have suggested that it is also present in vertebrates, including humans, and that its levels changed upon stress exposure, as well as in tumour cells and tissues. Many difficulties in accurately detecting this rare modified DNA base in mammals have however made its study tedious, and in order to further understand the biological importance of N6MeA, more straightforward detection methods are needed. The goal of this project was to develop a highly selective chemical reaction to modify N6MeA in DNA strands, to be used as basis for deploying novel reliable detection and mapping techniques. In a collaborative effort with the group of Prof. Matthew Gaunt here at the University of Cambridge, we have developed a new chemical reaction to selectively functionalise N6MeA in DNA strands and showed that this can be used for enriching DNA containing this modified base. We expect that this chemistry will have a considerable impact in the field, as has been the case for several other chemistry-based methods to manipulate modified DNA bases. We are currently expanding our efforts to apply this chemistry in different approaches to detect and map N6MeA.
Data: CORDIS, © European Union
Project objective
Herein, I propose to develop and adapt chemistry for the selective modification and tagging of N6-methyladenine (N6MeA) in the context of a DNA strand. Two promising chemical strategies will be applied, for which I have already established proofs-of-concept of efficiency and selectivity on DNA monomers (2'-deoxynucleosides and nucleotides). Once such a specific chemical labelling protocol has been optimised, I will use it to map N6MeA in genomic DNA with two different approaches: 1) by chemical pulldown of N6-methylated DNA fragments, sequencing of the enriched fragments, and alignment to a reference genome to generate a low-resolution N6MeA map. 2) By analysing the influence of the introduced tags and modifications on the PCR outcome and take advantage of their stalling of polymerases.The expected outcome is a first chemistry-assisted mapping of N6MeA. As for the chemical tagging of 5-formylcytosine (5fC) and 5-hydroxymethyluracil (5hmU), or oxidative and reductive bisulfite sequencing to sequence 5fC and 5-hydroxymethylcytosine (5hmC), all developed in the proposed host lab, it is expected to have a significant impact in the field. It will considerably facilitate the detection and mapping of genomic N6MeA in various species including mammals and humans, which is of tremendous importance to unravel the biological role of this DNA modification and identify novel biological as well as possible pathological pathways.
Original text from CORDIS.
Participants
- THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE · CAMBRIDGECoordinatorUnited Kingdom
Links
Data: CORDIS, © European Union