Single Molecule Measurements of the Unfolding Behavior of Diverse DNA Hairpin Assemblies

Abstract

Kinetic and thermodynamic effects of small structural changes to the DNA duplex can be studied on a single molecule level using optical tweezers. Short DNA hairpins, which can be synthesized to contain any sequence or structure of interest, serve as our model for double stranded DNA duplexes. To facilitate pulling with optical tweezers, the hairpins are attached to long flanking DNA “handles,” which themselves are chemically attached to beads. The forces required to fully unfold the hairpin constructs are directly related to the thermodynamic stability of the hairpin structure, allowing us to quantify the effects of mismatches, lesions, intercalator binding, and other non-canonical structures that disturb the stability of double-stranded DNA. The first step in this project is to synthesize, purify, and characterize hairpin assemblies with specific sequences, utilizing a combination of solid-phase organic synthesis and molecular biology. Then, individual DNA hairpin molecules are stretched with optical tweezers. Changes in distance and force reveal that fully-matched Watson-Crick duplex hairpins unfold and refold neatly in discrete, concerted events, whereas mismatched and bubbled hairpins open and close with lower force and greater force variability. Although the overall stability of the structures is predicted well by mfold calculations, the single molecule studies allow measurement of the barrier to unfolding as well as the distance to the transition state. After validating the method for DNA alone, changes in the transition state and hairpin stability in the presence of DNA binding ligands can then be probed directly.