Non-B conformations of CAG repeats using 2-aminopurine

Repetition of trinucleotide sequences is the molecular basis of ~30 hereditary neurological and neurodegenerative diseases, and alternate structures adopted by these sequences are implicated in the etiology of such diseases. Elucidating these structures is important for advancing mechanistic understanding and ultimately treatment. Studies of (CAG) repeats are motivated by their involvement in a number of these diseases, and the structures favored by (CAG)₈ are discussed in this contribution. Utilizing the strong effect of base stacking on fluorescence quantum yield, 2-aminopurine is used in place of adenine to determine the secondary structures adopted by such repeated sequences. Alone, (CAG)₈ folds into a hairpin comprised of a duplex stem and a single-stranded loop. Energetic studies indicate that the hairpin is anchored by the interactions in the stem and has a strained loop environment. As a model for intermediates that form during repeat expansion, (CAG)₈ was also incorporated into a duplex to form a three-way junction. In contrast to the isolated (CAG)₈, this integrated repeat adopts an open, unfolded loop. Enthalpy and entropy changes associated with denaturation indicate that the stability of the three-way junction is dominated by interactions in the duplex arms and that the repeated sequence tracks global unfolding. Because 2-aminopurine provides both structural and energetic information via fluorescence and also is an innocuous substitution for adenine, significant progress in elucidating the secondary structures of (CAG) repeats will be achieved.