Molecular Crowding Effects on Stability and Kinetics of Trinucleotide Repeat Hairpins

Abstract

Monovalent salt shifts the equilibrium toward the hairpin conformation by slowing the opening transition. In contrast, molecular crowding by PEG accelerates both the opening and closing transitions.The experiments examined the conformational changes of trinucleotide repeat-containing DNA hairpins that occur through formation and melting of intramolecular base pairs. The hairpins can exist in two forms: an open (unpaired) state and a closed (base paired) state ( Figure 1). Single-molecule fluorescence resonance energy transfer (smFRET) provides a powerful approach to monitor directly which conformation the hairpins are populating. For this method, the hairpins were constructed from two synthetic strands of DNA, each labeled with a different fluorescent molecule -Cy3 and Cy5. These fluorescent molecules comprise a FRET donor-acceptor pair. For this approach, the Cy3 dye is directly excited, and Cy3 transfers energy to Cy5 in a distance-dependent manner. The relative amounts of emitted light by Cy3 and Cy5 are correlated to the distance between the dyes, which can then serve as a signal for whether the hairpin is in the open or closed state. An individual hairpin emits primarily light corresponding to Cy5 in the closed state and to Cy3 in the open state. This setup provides a discrete signal for each conformation. Using widefield total internal reflection microscopy (TIRFM), discrete dye signals data are acquired from hundreds of individual molecules simultaneously. ABSTRACT OVERVIEW OF TRINUCLEOTIDE REPEAT DISORDERSNumerous 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) 5 and (CTG) 5 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) 5 and (CTG) 5 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 deleterio...