Chemical and Enzymatic Methods to Detect Chemical Modifications in Nucleic Acids

The bases of DNA and RNA are chemically modified in a variety of ways across all branches of life. DNA and RNA chemical modifications function as a layer of epigenetic information that regulate gene expression. Studies on the chemical reactivity of nucleic acids and the biochemical activity of enzymes that modify nucleic acids have created a biochemical toolbox with which we can study these epigenetic features to understand their function and distribution in normal biology and disease. The expansion of this biochemical toolbox through the development of new chemical methods to manipulate specific modified bases or through the innovation of enzymatic approaches is therefore of significant value. Chapter 2 outlines the development of a chemical approach to install a pyridine group selectively onto 5-methylcytosine. The pyridination reaction was a xanthone-photosensitised process involving 4-cyanopyridine that introduces a 4-pyridine modification at the methyl group of 5-methylcytosine. The transformation was compatible with a range of 4-cyanopyridine substrates. The introduced pyridine group can therefore function as a scaffold to introduce chemical groups to 5-methylcytosine that facilitate its manipulation and detection. In Chapter 3, 5mC pyridination was explored in DNA to determine whether a chemical group could be introduced to enable base resolution detection of 5mC by catalysing a specific 5mC-to-T conversion. In Chapter 4, cyanopyridines containing chemical enrichment handles were synthesised and tested to enable the chemical enrichment of m5C in RNA. A variety of enzymes characterised to modify genomic features are used for the detection of epigenetic features. One example is the use of Tn5 transposase in CUT&Tag. Chapter 5 describes the development of a method to covalently recruit Tn5 transposase to specific features in DNA.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Simpson, Mathew

Advisor dc:contributor.advisor

• Balasubramanian, Shankar

• Nucleic Acid Modifications

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/1a5d3c44-d78d-4bf4-8372-e7da826d144f/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.101966

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358180