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

Abstract

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. Temperaturebased, 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.

Conformational and migrational dynamics of slipped-strand DNA three-way junctions containing trinucleotide repeats

“…SM-FRET has been used previously to study the dynamics of isolated hairpins containing CAG or CTG repeats. Hairpins with 6-12 repeats of CAG or CTG separated by a polyA or polyT linker showed hairpin opening and closing transitions between the fully paired conformations, with CTG structures being more stable 35 . While short slip-outs could shift by the opening and closing of only a few base pairs 11 , this is increasingly unlikely as the number of repeats increase.…”

DNA hybridisation kinetics using single-molecule fluorescence imaging

“…In terms of hybridisation, the increased k on in the presence of cations is due to increasing the proportion of successful binding events [31] as the cations alter the structure of the ssDNAs making hybridisation more favourable [8]. This trend in increasing k on , however, is not seen for structured ssDNAs such as hairpins [31], as the opening rate of hairpins slows with increasing salt concentrations due to stabilisation of the secondary structure [67]. Although it is clear that an increase in salt concentration has a positive effect on k on for unstructured ssDNAs, any correlation with k off is not.…”

Long-Range Hairpin Slippage Reconfiguration Dynamics in Trinucleotide Repeat Sequences

“…However, we did not capture any blunt-end configuration state (E FRET ∼ 0.84) in the (CAG) n repeats we probed. Possibly, the CAG•CAG antiparallel interaction is too weak to hold an unstable 3-nt CAG loop stably, 32 resulting in a much energetically unfavored 7-nt dAGCAGCA loop. In the evennumber cases, the blunt-end configuration consists of a longer stem (with an additional C•G pair) and a more stable tetranucleotide TGCT (or AGCA) loop compared with the overhang counterpart.…”