Summary
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.
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.
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More information & hyperlinks
| Web resources: | https://cordis.europa.eu/project/id/789407 |
| Start date: | 19-04-2018 |
| End date: | 18-04-2020 |
| Total budget - Public funding: | 183 454,80 Euro - 183 454,00 Euro |
Cordis data
Original description
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.
Status
CLOSEDCall topic
MSCA-IF-2017Update Date
28-04-2024
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