Three-way helical junctions (3WJs) arise in genetic processing, and they have architectural and functional roles in structured nucleic acids. An internal bulge at the junction core allows the helical domains to become oriented into two possible, coaxially stacked conformers. Here, the helical stacking arrangements for a series of bulged, DNA 3WJs were examined using ensemble fluorescence resonance energy transfer (FRET) and single-molecule FRET (smFRET) approaches. The 3WJs varied according to the GC content and sequence of the junction core as well as the pyrimidine content of the internal bulge. Mg²⁺titration experiments by ensemble FRET show that both stacking conformations have similar Mg²⁺ requirements for folding. Strikingly, smFRET experiments reveal that a specific junction sequence can populate both conformers and that this junction undergoes continual interconversion between the two stacked conformers. These findings will support the development of folding principles for the rational design of functional DNA nanostructures.

Leveille, M.P., T. Tran, G. Dingillo, and B. Cannon. Detection of Mg2+-dependent, coaxial stacking rearrangements in a bulged three-way DNA junction by single-molecule FRET. Biophysical Chemistry. 2018. In press.

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.

Mitchell, M.L., M.P. Leveille, R.S. Solecki, T. Tran, and B. Cannon. Sequence-Dependent Effects of Monovalent Cations on the Structural Dynamics of Trinucleotide-Repeat DNA Hairpins. J. Phys. Chem. B. 2018. In press.