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Development of chemical methods for DNA N6-methyladenine mapping

Periodic Reporting for period 1 - N6MeA ChemSeq (Development of chemical methods for DNA N6-methyladenine mapping)

Reporting period: 2018-04-19 to 2020-04-18

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.
A major part of the work consisted of identifying a viable approach for the chemoselective functionalisation of N6MeA under DNA-compatible condition. Upon extensive investigation of the reactivity of this modified base, we ultimately developed a completely new reaction that allowed for the selective functionalisation of N6MeA in short DNA strands.

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,
The thorough investigation of the reactivity of N6MeA led to the identification of a chemical process addressing the challenging task of chemoselectively functionalising this chemical entity under DNA-compatible conditions. This exemplifies that mild and water-compatible chemical processes can be found for the selective manipulation of specific chemical features deemed rather unreactive in biomacromolecules. By using an appropriate probe compound that we designed to be compatible with this chemical functionalisation, the modular tagging of N6MeA in DNA strands is possible. This chemistry lays the basis for the development of different applications that have the potential to facilitate the study of this modified DNA base. To demonstrate the utility and potential for application of the developed tagging approach, we have developed a chemical enrichment protocol to enrich for N6MeA-containing DNA strands. This has allowed us to confirm that our chemistry is applicable on large single-stranded and double-stranded DNA strands and represents a proof-of-concept for the potential application of an N6MeA mapping technique.

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.