Contrasting Effects of Molecular Crowding on Trinucleotide-Repeat DNA Hairpins

Trinucleotide-repeat motifs are associated with numerous disorders, and genetic instability arising from these repeat motifs may be related to their propensity to form nonhelical structures. Genetic processing and organization of repeat-containing domains occur within a molecularly crowded environment. To address the influence of molecular crowding on structure formation by trinucleotide-repeat motifs, a single-molecule FRET, DNA hairpin system reveals contrasting effects of crowding on hairpin formation for two different trinucleotide-repeat sequences, CAG and CTG. Molecular crowding causes structure formation by the repeat domains to become more dynamic, with sequence-dependent effects arising from differences in the central mismatch of the trinucleotide-repeat motifs. Crowding conditions promote hairpin formation by accelerating the hairpin closing transition; however, these conditions can also weaken the hairpin structure through crowder size-dependent acceleration of the hairpin opening transition. Molecular crowding shifts the conformational equilibria for repeat sequences away from the hairpin state and toward the unstructured state with increasing NaCl. Temperature-based, single-molecule FRET experiments indicate that molecular crowding has entropic and enthalpic contributions to the folding pathways of these molecules. Molecular crowding can perturb DNA structure formation by mismatch-rich repeat domains and may have an impact on genetic instabilities and their pathological outcomes.