Equilibrium and irreversible unzipping of DNA in a nanopore

Abstract

We study the unzipping dynamics of individual DNA hairpins using nanopore force spectroscopy at different voltage ramp rates and temperatures. At high ramp rates the critical unzipping voltage is proportional to log ˙ V ,w here˙ V is the voltage ramp. At low ramp values we observe a crossover to another regime with a weaker dependence on ˙ V. Here we report on the dependence of these two regimes on temperature. Remarkably, the unzipping kinetics can be well described by a simple two-states model that predicts the existence of two asymptotic regimes: quasi-equilibrium unzipping at low-voltage ramps and irreversible unzipping at high ramp rates. Introduction. - The dynamics of DNA unzipping and re-zipping is centralfor many biological processes such as replication, transcription and DNA repair (1). The thermodynamic properties of base-pairing in DNA were determined by equilibrium measurements in bulk, and more recently single-molecule methods were applied to the study of basepair unzipping kinetics, and of the forces that enzymes apply in order to pull apart the two strands (2-7). Nanopore force spectroscopy is an emerging single-molecule technique that allows one to test thousands of copies of the same molecule in short period of time (8); unlike other single- molecule methods the polynucleotides do not need to be grafted to macroscopic beads or to surfaces. Instead, an electric field is used to guide the charged biopolymer through a narrow pore (nanopore) that can only admit a single strand of DNA (ssDNA) at a time. The toxin α-Hemolysin (α-HL) has been extensively used to study the translocation dynamics of unstructured polynucleotides (9-11) and to unzip short double stranded DNA molecules (8, 12, 13). Voltage drop across the nanopore applies force on the molecule in order to unzip it, and the unzipping time is precisely measured by the simultaneous recording of the residual ion current that flows through the blocked pore. Recently, we demonstrated that a dynamic voltage scheme can be used in this experiment to decouple the initial entry process of ssDNA overhang into the pore, from the subsequent unzipping kinetics. This gave us access to very small forces or small loading rates, and in particular to unzipping voltages much below the threshold potential required for the entry of the ssDNA to the pore (∼ 50 mV). At high volt- ages or high loading rates (defined as the change in voltage over time, ˙