Abstract
Post-transcriptional gene regulation comprises a range of mechanisms used by a cell to control the expression of RNA molecules in response to environmental changes. These native mechanisms can be exploited and modified to produce synthetic molecular switches for the control of enzyme expression. One such synthetic mechanism is that of antisense oligonucleotides, whereby modified antisense oligos (such as LNA gapmers) bind mRNA targets and mediate mRNA blocking or cleavage. This is done by binding the ribosome binding site (RBS) and halting ribosome progression or by mediating cleavage of the RBS via ribonucleases.Due to the vast number of nucleic-acid interactions that can result in up or down regulation of gene expression, a high throughput method of testing is essential. Here we establish a novel, high throughput RNA array-based assay that can be used to optimise and refine LNA gapmers as synthetic molecular OFF switches. Additionally, this assay has been modified to increase accessibility with readily available low-throughput techniques. Moreover, an RNA array-based substrate array, with potential high throughput capability, has been developed that can measure enzyme expression and subsequent enzyme activity. This novel assay utilised high resolution surface microscopy techniques to measure solid substrate degradation.
It is anticipated that the novel RNA-array based assays developed here could be used to test libraries of candidate synthetic molecular switches as a refinement tool. Similarly, the RNA-array based substrate array could be used to test enzyme variant expression and activity.
| Date of Award | 30 Apr 2024 |
|---|---|
| Original language | English |
| Awarding Institution |
|
| Supervisor | Anastasia Callaghan (Supervisor), Garry Scarlett (Supervisor) & Darren Gowers (Supervisor) |