| Studies of single molecules provide important distribution and dynamic information that can be lost by ensemble averaging in other techniques. The ability to measure the kinetics and biophysical properties of DNA unzipping at the single molecule level will be very important in molecular biology, illuminating for example the timescale and forces that individual proteins could access the single stranded DNA template for transcription. Herein is described a technique to measure the unzipping kinetics of double-stranded DNA by pulling one strand of the duplex through alpha-hemolysin in a standard patch-clamp apparatus. An optimized PCR protocol yielded the first direct proof of strand separation in such a system. The time to unzip each molecule could be inferred from the ionic current signature of DNA traversal. A kinetic model was derived from the unzipping time distribution. The effects of inserting base pair mismatches, and of varying the applied voltage, the temperature, and the ionic strength of the solution used to unzip the DNA are analyzed within the kinetic model. The rate constants and enthalpy barriers to the unzipping reaction are calculated. Furthermore, the effective charge of a nucleotide in the protein pore is estimated to be q ∼ 0.1 e. This estimate of the effective charge is important both because it sheds light on the behavior of screening in the confined environments and because it markedly improves the basic understanding of a system, the alpha-hemolysin pore, that has been the focus of substantial biotechnological activity. In contrast to other micromechanical single-molecule techniques, this new method is particularly well suited to study the unzipping behavior of oligomers (<200 bp). Because shorter strands of DNA can be chemically synthesized, the nanopore can be used to study molecules of any desired sequence and length. Here, the method was used to study the unzipping of various lengths of homopolymers (poly dA-dT), revealing an unexpectedly large influence of end effects. The technique is discussed both for its potential as a new method to study single molecule interactions and in the context of an emerging technology for single molecule ultra-fast DNA sequencing. |
