Sticky DNA: In Vivo and In Vitro Association of Long GAA·TTC Tracts to Generate Two Independent Supercoiled Domains

Abstract

The GAA·TTC trinucleotide repeat expansion associated with Friedreich's ataxia (FRDA) is prone to form non-B DNA structures, i.e. hairpins, triplexes and sticky DNA. We have explored the ability to reconstitute sticky DNA in vitro. Changing metal ion concentrations and negative supercoiling in the presence of plasmids harboring two (GAA·TTC)176 tracts enabled the formation of the sticky DNA structure. The model of sticky DNA was confirmed by the presence of two independent negatively supercoiled domains that are topologically constrained by the association of the two (GAA·TTC)176 tracts. The topological consequence of the sticky DNA conformation in vivo was shown to be a lower (less negative) average supercoil density compared to the other non-B DNA forming sequences. This was a unique observation seen only with sticky DNA forming plasmids. Long GAA·TTC repeats that can form non-B DNA structures were found to cause transcription inhibition both in vitro and in vivo. The loss of transcriptional activity and mRNA are critical steps in the etiology of FRDA. Through the binding of GAA-specific polyamide ligands to the DNA helix, the formation of the sticky DNA conformation was inhibited. Thus, the binding of the ligands to the DNA shifts the equilibrium from the non-B structure to the traditional B-DNA and therefore alleviates the transcription inhibition. Hence, these findings support the concept of developing therapeutics for the treatment of FRDA. [R. Burnett, C. Melander, J. Puckett, L. Son, R.D. Wells, P. Dervan and J. Gottesfeld, in preparation]. Supported by NIH and FARA.