Molecular Crowding Effects on Stability and Kinetics of Trinucleotide Repeat Hairpins

Abstract

Numerous genetic disorders arise from the propensity of certain repetitive DNA sequences to form intrastrand, non-helical structures, such as hairpins, due to extensive self-complementarity. Within the cellular environment, the formation of these non-helical structures occurs within highly crowded conditions. Using single-molecule FRET microscopy, the effect of molecular crowding on the stability and dynamics of individual DNA hairpins containing the trinucleotide repeat sequences (CAG)N and (CTG)N were explored. These repeat sequences have central mismatches within each repeat unit that reduce the stability of intrastrand contacts between repeat units in the stem, leading to highly dynamic behavior for their hairpins. The structural dynamics for (CAG)N and (CTG)N trinucleotide repeat hairpins were quantified in the presence of molecular crowding agents (polyethylene glycol; PEG) with different sizes. Analysis of the transitions rates between the unstructured and closer conformations shows that an increase in molecular crowding promotes the formation of the hairpins. Strikingly, the crowding agents induce a greater acceleration of hairpin melting and overall destabilization, which contrasts with observations from controls with fully paired DNA hairpins. Under these conditions, the high mismatch content of these repeat hairpins becomes more destabilizing in the presence of PEG. The findings indicate that molecular crowding may confer some protective benefit to genomic DNA by disrupting these deleterious structures at smaller repeat sizes.