Intrastrand Base Pair Formation in Repetitive DNA Sequences

Abstract

Repetitive trinucleotide DNA motifs at distinct genetic loci are responsible for triggering a broad group of hereditary, neurodegenerative diseases. The occurrence of non-helical DNA structure within these motif-harboring regions has long been identified as a structural precursor that interferes with normal DNA processing and ultimately leads to pathogenic states. The precise role of these structures in this interference remains unresolved; therefore, an improved understanding of these structures is critical for developing targeted therapies. As a first step, we characterized the formation and dynamics of CAG and CTG repeat DNA hairpins, a specific non-helical structure that can form within these trinucleotide sequences, which are associated with numerous forms of spinocerebellar ataxia and Huntington's disease. DNA constructs were designed that contain a specific number of either CAG or CTG repeats, and they were labeled at specific sites with the fluorescent dyes Cy3 and Cy5. Using single-molecule fluorescence microscopy, we directly imaged nanoscale changes in distance between the fluorescence dyes for individual DNA molecules in real time with sub-second resolution. The changes in interdye distance arise due to continuous conformational transitions between two hairpin states (open and closed). The time traces revealed that the CTG repeats form more stable hairpins than the CAG repeats. Strikingly, both sequences exhibit highly dynamic behavior with rapid open and closing rates. In addition, increases in the monovalent salt concentration increased the likelihood of the hairpin in the closed conformation. Analysis of the open and closing behavior revealed two reasons for this observation: 1) the closed form of the hairpin was longer lived at higher salt concentrations and 2) the transition from the open to the closed state occurred faster.