DNA translocation and diffusion
through the protein pore a-Hemolysin
DNA molecules can be electrophoretically threaded through nanometer scale pores ("nanopores") in a process named "DNA translocation." If the nanopore diameter is smaller than twice the DNA cross section (~2 nm for single-stranded DNA or ~4 nm for double-stranded DNA) the DNA molecules must progress through the pore in an unfolded, single file manner.
What are the forces and interactions that govern DNA translocations? Does this process depend on the DNA structure and sequence? Fortunately, the dynamics of DNA translocation can be directely measured, at the single molecule level. When the DNA enters the pore, it gives rise to an abrupt drop in the ionic current flowing through the pore. This signal is used to detect the residence time of the biopolymers in the pore, see Figure 1.
We find that the translocation time depends strongly on the DNA sequence: the translocation of purines are roughly three fold longer than that of pyrimidines. Figure 2 is an "event diagram" showing more than 6,000 individual DNA translocation events, represented by their duration (tT) and blocked current. Here we compare same length, poly-Cytosines (blue), poly-Adenines (red) and alternating Adenine-Cytosine ssDNA (green). Two salient features can be seen: 1) The translocation times of the Adenines are significantly longer than the cytosines (roughly a factor of 3). 2) The Adenine's times are far more broadly distributed as compared with the Cytosines, or the alternating Cytosines-Adenines. These results indicate that the purines have stronger interactions with the nanopore giving rise to much longer translocation times. The broad dispersion in the Adenines is due to purines strong self-stacking interactions, which should be partly resolved before translocation is possible.
Using a real time Dynamic Voltage Control method, we can change the voltage applied on the DNA while it is occupying the pore. This method allows us to extend the range of voltages that can be used to probe DNA translocations, and to apply time-varying voltage profiles. In order to enter the pore, the biopolymer has to overcome an energy barrier (roughly 12 kBT for the alpha-Hemolysin). This barrier is reduced when a larger voltage is applied. Thus the entry rate, to a first approximation, depends exponentially on the voltage. Dynamic Voltage Control allows us to probe small voltages, in which the entry rate is extremely small (minutes or hours between events). In particular, we can set the voltage to zero, and measure the time distributions of unbiased DNA diffusion from the nanopore, or an assisted diffusion (diffusion + small drift). Read more on that in Ref. 5.
- Meller, A., L. Nivon, E. Brandin, J. Golovchenko and D. Branton. (2000) Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl. Acad. Sci. USA. 97, 1079-1084.
- Meller, A., L. Nivon and D. Branton. (2001) Voltage-Driven DNA Translocations through a Nanopore. Phys. Rev. Lett. 86, 3435-3438.
- Meller, A. and D. Branton. (2002) Single molecule measurements of DNA transport through a nanopore. Electrophoresis 23, 2583-2591.
- Branton, D. & A., Meller. (2002) Using nanopores to discriminate between single molecules of DNA (in "Structure and dynamics of confined polymers", eds. J.J. Kasianowicz & D.W. Deamer) 177-185, Kluwer Academic Publishers.
- Meller, A. (2003) Dynamics of polynucleotide transport through nanometre-scale pores. J. Phys.: Condens. Matter 15, R581-R607.
- Bates, M., M. Burns and A. Meller. (2003) Dynamics of single DNA molecules actively controlled inside a membrane channel. Biophys. J. 84(4), 2366-2372.
- Mathé, J., Aksimentiev, A., Nelson, D.R., Schulten, K. & Meller, A. (2005) Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel. Proc Natl Acad Sci U S A 102, 12377-12382.
- Jan Bonthuis, D., Zhang, J., Hornblower, B., Mathe, J., Shklovskii, B.I. & Meller, A. (2006) Self-Energy-Limited Ion Transport in Subnanometer Channels. Phys. Rev. Lett. 97, 128104 -128101.
- Wanunu, M., Chakrabarti, B., Mathé, J., Nelson, D.R. & Meller, A. (2008) Orientation Dependent Interactions of DNA with an α-Hemolysin Channel, Phys. Rev. E, 77, 031904.
- Wanunu, M. & Meller, A. (2008) Single Molecule Analysis of Nucleic Acids and DNA-protein Interactions using Nanopores. (in "Laboratory Manual on Single Molecules", eds. T. Ha & P. Selvin), Vol. 395-420. Cold Spring Harbor Press.