A photochemical method for the detection of cytosine modifications in DNA

Abstract

Covalent modifications to DNA bases represent an important form of epigenetic information which contributes to the regulation of gene expression in many cellular processes throughout development and differentiation. Aberrant modification patterns can also signal or drive the emergence of genetic disease states. Techniques that enable non-canonical bases to be detected and mapped throughout the genome are therefore of academic and diagnostic interest. To this end, a toolbox of chemical and enzymatic methods has been developed to selectively manipulate the unique functionalities of covalent DNA modifications; however, bisulfite sequencing, the benchmark technique for the detection of 5-methylcytosine and its oxidised derivatives, is highly destructive and greatly reduces the sequence complexity of DNA samples. These limitations are being addressed through the development of bisulfite-free sequencing methods that seek to detect modified DNA bases under non-degrading conditions. This thesis describes the discovery and development of a photochemical treatment that alters the hydrogen bonding pattern of 5-carboxycytosine to a uracil analogue. The photochemical conversion is selective for the unique carboxylate functionality of 5-carboxycytosine over other canonical and modified bases. The reaction was first identified in experiments on nucleoside monomers, before being optimised in model oligonucleotides and genomic DNA fragments. The transformation was investigated using experimental, physical and computational techniques and a plausible mechanistic pathway was proposed. The conversion successfully enables the detection of 5-carboxycytosine within a modified oligonucleotide and, in conjunction with enzymatic oxidation by TET, the detection of 5-methylcytosine in a model modified genome via next-generation sequencing. The mild irradiation with visible light in aqueous conditions is well-suited to the manipulation of complex biological macromolecules such as nucleic acids, and this reaction represents the first example of a photochemical method for profiling cytosine modifications at single-base resolution.