Sequence-Dependent Effects of Monovalent Cations on the Structural Dynamics of Trinucleotide-Repeat DNA Hairpins

Repetitive trinucleotide DNA sequences at specific genetic loci are associated with numerous hereditary, neurodegenerative diseases. The propensity of single-stranded domains containing these sequences to form secondary structure via extensive self-complementarity disrupts normal DNA processing to create genetic instabilities. To investigate these intrastrand structural dynamics, a DNA hairpin system was devised for single-molecule fluorescence study of the folding kinetics and energetics for secondary structure formation between two interacting, repetitive domains with specific numbers of the same trinucleotide motif (CXG), where X = T or A. Single-molecule fluorescence resonance energy transfer (smFRET) data show discrete conformational transitions between unstructured and closed hairpin states. The lifetimes of the closed hairpin states correlate with the number of repeats, with (CTG)(N)/(CTG)(N) domains maintaining longer-lived, closed states than equivalent-sized (CAG)(N)/(CAG)(N) domains. NaCl promotes similar degree of stabilization for the closed hairpin states of both repeat sequences. Temperature-based, smFRET experiments reveal that NaCl favors hairpin closing for (CAG)(N)/(CAG)(N) by preordering single-stranded repeat domains to accelerate the closing transition. In contrast, NaCl slows the opening transition of CTG hairpins; however, it promotes misfolded conformations that require unfolding. Energy diagrams illustrate the distinct folding pathways of (CTG)(N) and (CAG)(N) repeat domains and identify features that may contribute to their gene-destabilizing effects.