Boston University

Nanopore Force Spectroscopy - Application to Unzipping of DNA duplexes

Many cellular processes, such as DNA replication and RNA transcription, involve the unzipping of double-stranded DNA by proteins. The forces and time scales associated with the breakage of the bonds, which stabilize the secondary and tertiary structures of nucleic acids, can now be studied at the single molecule level (SM). SM analysis of biomolecules can reveal information masked by ensemble averaging, such as the variation in the behavior of putatively identical molecules, and short lived intermediate states.

We have developed a nanopore based method to apply a time dependent force on individual DNA or RNA molecules, or on nucleic acids-protein complexes. Our method can be used to control the unzipping kinetics of individual DNA hairpins at a constant force as well as at a constant loading rate. Furthermore, because our method does not entail the physical coupling of the test molecules to a force transducer, very high throughput can be achieved, exceeding ~100 molecules per minute. Figure 1 displays a schematic movie of DNA hairpin unzipping when threaded through the ~1.5 nm alpha-Hemolysin pore.

Each nanopore unzipping event consists of: 1. Threading and sliding the single stranded DNA hairpin overhang, typically at 120 mV. 2. Holding the DNA inside the pore for a brief period of time using a small voltage. 3. Unzipping of the DNA at a constant voltage ramp. See Figure 2 for a typical voltage profile used in the experiments. The unzipping moment is signaled by a jump in the current flowing through the pore during the ramp period, as shown in Figure 3. Distributions of the unzipping voltages of typically 1,000 separate unzipping events at each given ramp value are automatically obtained.

We use our method to extend the range of force accessible to other techniques to study DNA unzipping kinetics. We find that the static unzipping times decrease exponentially with voltage with a characteristic decay constant that is independent of the duplex region sequence. Our dynamic unzipping measurements (time-varying force) are in agreement with the ~log(v) dependence at high v (where v is the loading rate or “velocity”) as observed by other methods. An extension of our measurements to lower v levels reveals a weaker dependence on log(v) as shown in Figure 4. In this regime the hairpin can rebind quickly before it is ejected from the pore, giving rise to the weaker dependence on v.

A recent paper by Dudko, Hummer and Szabo (see Ref #1) demonstrated a remarkably simple method to directly determine force-dependent lifetimes from rupture force histograms, callected at different pulling voltage ramps. The model was validated and implemented using our nanopore force spectroscopy data (Figure 5 ), showing a remarkable agreement between constant voltage (force) and constant ramp unzipping experiments.


  1. Dudko, O., Hummer, G. and A. Szabo (2008) Theory, analysis and interpretation of single-molecule force spectroscopy experiments. Proc. Natl. Acad. Sci. 105, 15755-760.
  2. Dudko, O., J. Mathé, A. Szabo, A. Meller and G. Hummer (2007) Extracting kinetics from single-molecule force spectroscopy: Nanopore unzipping of DNA hairpins. Biophys. J., 92, 4188-4195
  3. Mathé, J., H. Visram, V. Viasnoff, Y. Rabin and A. Meller. (2004) Nanopore unzipping of individual DNA hairpin molecules. Biophys. J. 87(5), 3205-3212.
  4. Mathé, J., A. Arinstein, Y. Rabin and A. Meller. (2006) Equilibrium and irreversible unzipping of DNA in a nanopore. Europhys. Lett. 73(1), 128-134


National Science Foundation

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