Synthesis of nucleoside analogues and peptides for nanoore analysis and controlled bioactivity

This research describes the synthesis of nucleoside analogues and peptides, and their application to nanopore technology and cell biology. Nanopore technology is a technique in which analytes are detected by disturbances in ionic current caused as they traverse a pore, at a fixed applied potential. Here potential applications of the technology are explored. A nanopore-based method to detect enzymatic activity was investigated. The technique involved the detection of peptide fragments proteolytically released from a solid-support. The released fragments gave rise to distinct electrical signals which facilitated their identification and characterisation. The frequency with which they traversed the pore was indicative of enzyme activity. The technique was successfully applied to the detection of the protease enzyme, renin, in the presence of human serum. An approach to count nucleotide repeat sequences, which are the basis of forensic DNA fingerprinting, was also investigated. It was hypothesised that chemically modified copies of the repeat regions could be generated using primer extension and nucleoside analogues. The copying could be performed such that a single analogue was incorporated per repeat. Ensuing nanopore analysis would then give rise to electrical signals in which the analogues could be identified from unique signals. The number of repeat sequences could simply be determined by counting the number of signals. It was envisaged that this could be achieved using adamantane-modified nucleosides. However, these did not give rise to the expected result. Inspired by the adamantane project, analogues of the drug, azacytidine were synthesised. Azacytidine is used to treat cancers that arise due to epigenetic modifications of DNA. It was hypothesised that its side-effects could be mitigated using photocaged-derivatives of the nucleoside, as these would enable greater temporal and spatial control of drug release. Analogues of azacytidine were successfully synthesised and shown to be uncaged back to the parent nucleoside

A Photo‐responsive Small‐Molecule Approach for the Opto‐epigenetic Modulation of DNA Methylation

Controlling the functional dynamics of DNA within living cells is essential in biomedical research. Epigenetic modifications such as DNA methylation play a key role in this endeavour. DNA methylation can be controlled by genetic means. Yet there are few chemical tools available for the spatial and temporal modulation of this modification. Herein, we present a small‐molecule approach to modulate DNA methylation with light. The strategy uses a photo‐tuneable version of a clinically used drug (5‐aza‐2′‐deoxycytidine) to alter the catalytic activity of DNA methyltransferases, the enzymes that methylate DNA. After uptake by cells, the photo‐regulated molecule can be light‐controlled to reduce genome‐wide DNA methylation levels in proliferating cells. The chemical tool complements genetic, biochemical, and pharmacological approaches to study the role of DNA methylation in biology and medicine