Nanopore Unzipping of Individual DNA Hairpin Molecules

Abstract

We have used the nanometer scale alpha-Hemolysin pore to study the unzipping kinetics of individual DNA hairpins under constant force or constant loading rate. Using a dynamic voltage control method, the entry rate of polynucleotides into the pore and the voltage pattern applied to induce hairpin unzipping are independently set. Thus, hundreds of unzipping events can be tested in a short period of time (few minutes), independently of the unzipping voltage amplitude. Because our method does not entail the physical coupling of the molecules under test to a force transducer, very high throughput can be achieved. We used our method to study DNA unzipping kinetics at small forces, which have not been accessed before. We find that in this regime the static unzipping times decrease exponentially with voltage with a characteristic slope that is independent of the duplex region sequence, and that the intercept depends strongly on the duplex region energy. We also present the first nanopore dynamic force measurements (time varying force). Our results are in agreement with the approximately logV dependence at high V (where V is the loading rate) observed by other methods. The extension of these measurements to lower loading rates reveals a much weaker dependence on V.

Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore

“…Let there be Ϸ30 nt in the entire lumen of the pore (approximately the same as there would be for a strand in a double helix 10 nm in length) and therefore Ϸ15 nt in the transmembrane ␤-barrel. The experimentally determined effective charge on each base is Ϸ0.1e (37,38). This low value is consistent with the theory of Zhang and Shklovskii (36).…”

DNA in Nanopores: Counterion Condensation and Coion Depletion

“…For example, as has been noted in reference [6], the theory overestimates (by more than an order of magnitude) the effective pulling force on the DNA. A similar discrepancy concerning the magnitude of the force acting on DNA has been observed in recent experiments on nanopore-induced opening of DNA hairpins [16]. Furthermore, while the models focus on the kinetics of DNA translocation through the pore, experiments probe this process only indirectly, by monitoring the transient blocking of the current of small ions through the channel.…”

Theory, analysis, and interpretation of single-molecule force spectroscopy experiments

“…We first validate the methodology by comparing the results for the unzipping of DNA hairpins in a nanopore to those obtained previously by using the full power of the maximum-likelihood method (24). In nanopore force spectroscopy, an applied electric field is used to drive a charged polymer such as DNA through a membrane channel (27,28). The unzipping events are detected by monitoring the increase in the ionic current through the pore.…”