Friedreich’s ataxia (FRDA) is a recessive inherited neurodegenerative disorder, characterised by a trinucleotide repeat expansion mutation on the FXN gene consisting of a (GAA)n base triplet, where n = 66-1700. This mutation results in the transcriptional silencing of the FXN gene. One hypothesis for how this mutation causes gene silencing is the formation of a persistent DNA, or DNA-RNA, triplex. The work presented herein focuses on the biophysical study of DNA and DNA-RNA triplexes formed from sequences containing the (GAA)n sequence of varying lengths, and subsequently the development of fluorescence lifetime probes with the aim to image triplexes in cellulo.
Initially, the formation and stability of triplexes made from short oligonucleotides containing the (GAA)n FRDA repeat sequence, where n = 5, 10 and 20, was studied under various pH and salt conditions. It was found that triplexes only formed under acidic conditions.
A molecular biology approach was taken to produce polynucleotides containing a (GAA)n FRDA repeat sequence of pathological length, where n = 55, 75 and 200. The formation of DNA-DNA and DNA-RNA hybrid structures under varying conditions was studied using gel electrophoresis and AFM. It was found that multiple higher order structures formed at acidic pH with DNA, and physiological pH with DNA-RNA. The DNA triplexes formed at acidic pH were visualised with AFM.
Lastly, fluorescence lifetime probes based on the structure of thiazole orange were synthesised, and their fluorescence properties when bound to different DNA topologies were investigated. It was found that all probes exhibited long fluorescence lifetimes when bound to triplex DNA, presenting a strategy for imaging triplexes. These probes were then incubated with cells containing a (GAA)310 mutation and the fluorescence lifetime was measured using FLIM. However, no difference in average lifetime was seen between these cells containing the mutation and the wild type.