Frustration Between Preferred States of Complementary Trinucleotide Repeat DNA Hairpins Anticorrelates with Expansion Disease Propensity

The expansion of DNA trinucleotide repeats (TRs) beyond a threshold often results in neurodegenerative diseases in humans. The mechanisms causing these expansions remain unknown, although the tendency of TR ssDNA to self-associate into hairpins that slip along their length is widely presumed to be related. Here we apply single molecule FRET (smFRET) experiments and molecular dynamics simulations to determine conformational stabilities and slipping dynamics for CAG, CTG, GAC, and GTC hairpins. By developing novel analysis approaches for states with closely spaced FRET efficiencies along with improved transition detection algorithms, we determined the kinetic slipping schemes for these hairpins. Tetraloops are favored in CAG (89%), CTG (89%) and GTC (69%) while GAC favors triloops. We also determined that TTG interrupts near the loop in the CTG hairpin stabilize the hairpin against slipping (as do CAA substitutions in CAG hairpins). The different loop stabilities have implications for intermediate structures that may form when TR-containing duplex DNA opens. Opposing hairpins in the (CAG) ∙ (CTG) duplex would have matched stability whereas opposing hairpins in a (GAC) ∙ (GTC) duplex would have unmatched stability. This unmatched stability would introduce mechanical stress or frustration in the (GAC) ∙ (GTC) opposing hairpins that would be absent in (CAG) ∙ (CTG) structures. Given the biological observation that the CAG and CTG TR can undergo large, disease-related expansion whereas the GAC and GTC sequences do not, the mechanical stability differences we have identified can inform and constrain models of the expansion mechanisms of TR regions.