Electrically directed gene expression (EDGE): using switchable DNA triplexes and electrolysis to modulate transcription in a cell-free medium

Abstract

External control of gene expression is a powerful tool for molecular biology that benefits both research and commercial applications. Being able to control genetic systems using electrical signals would enable more sophisticated control of biological activity. For instance, automated systems could activate or deactivate gene expression in response to changes, begin expression at a desired time point, finely control the rate of production, or express an exact quantity of product. To bridge the gap between electrical and genetic systems I have developed Electrically Directed Gene Expression (EDGE), a DNA nanoswitch approach to controlling transcription. These nanoswitches use pH-dependent DNA triple helices (triplexes) to inhibit T7 RNA polymerase.

The pH is controlled electrically using electrolysis, where the decomposition of water induces a shift in pH level. The pH can be shifted either up or down depending on the polarity of the current being applied. This change in pH and triplex formation can then be reversed by swapping the polarity and electrolysing again. In this thesis I demonstrate that sending the pH up or down electrically can form or destabilise triplexes, and that this can be used to control the level of gene expression. Gene expression was measured using a fluorescent RNA aptamer called iSpinach, and this project required the design and production of custom hardware that was capable of large scale electrolytic control.

The use of high-throughput automation platforms enabled the production of 48 different EDGE constructs and 48 corresponding non-triplex controls. This allowed for the testing of a large number of variable triplex design parameters. This work successfully demonstrated both the electrical control of triplex-mediated inhibition of transcription, and how alterations to triplex design can impact the dynamics of this electrical response.