Periodic Reporting for period 1 - N6MeA ChemSeq (Development of chemical methods for DNA N6-methyladenine mapping)
Período documentado: 2018-04-19 hasta 2020-04-18
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.
The next step was to design a compatible probe to functionalise N6MeA with a modular handle that would allow the use of established bioorthogonal chemistry to selectively install desired tags onto N6MeA. We developed a probe that can be installed onto N6MeA using the developed reaction. This can be used for downstream modular functionalisation, including the installation of a functional handle.
We then developed an effective strategy to pull down DNA strands that contain N6MeA. Our efforts ultimately led to a procedure to enrich for N6MeA-containing single-stranded as well as double-stranded DNA, even in the presence of excess background unmethylated DNA. This lays the basis for the development of new methods to map N6MeA in genomes.
The work is currently in the process of being published and a patent application is pending. Further efforts in dissemination will follow once the work is protected and published. More broadly, the importance and impact of developing chemical approaches to study modified DNA bases has been highlighted in a peer-reviewed perspective (A. Hofer, Z. J. Liu, S. Balasubramanian, J. Am. Chem. Soc. 2019, 141, 6420–6429), and an broad overview on modified DNA bases and chemical approaches for their study has been presented to a general audience in Cambridge UK at a Pint of Science event (May 2018, https://pintofscience.co.uk/event/a-night-of-nucleic-acids).
With chemistry having played a pivotal role for the study of modified DNA bases in the past, we anticipate that this novel chemistry with demonstrated potential for application will have a considerable impact in helping to unravel roles and relevance of N6MeA in genomes. We are currently deploying this chemistry for the detection and mapping of N6MeA, pursuing different approaches that will contribute to enlarge the sparse chemical toolbox to study this modified base in the foreseeable future.