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DNA translocation through nanopores

DNA molecules can be electrophoretically threaded through nanometer scale pores ("nanopores") in a process called "DNA translocation." We distinguish between two main types of nanopores: a) protein pores, such as the toxin alpha-Hemolysin, and b) synthetic nanopores made in either organic or nonorganic films. 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) then the DNA molecules must progress through the pore in an unfolded, single file manner. In contrast, if the pore diameter is larger than twice the DNA diameter, the DNA can enter in a folded state.
When the DNA enters the pore, it induces 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, defined as the 'translocation time' (see Figure 1).

Translocation of single-stranded DNA
The translocation time of single-stranded DNA is strongly affected by its sequence. Specifically, pyrimidines(Thymines or Cytosines) translocate almost 3-fold faster than purines (Adenines or Guanines) . Figure 2 displays a comparison between poly-Cytosines (blue) and poly-Adenines (red) on an "event-diagram" wherein each event is represented by its duration (tT) and blocked current. Notably, the blue cluster of events is centered around much shorter times than the red cluster (these polymers have the same exact length). A biopolymer, which is composed of alternating Adenines and Cytosines,forms a cluster of events that fall in between the Adenines and Cytosines translocation times (green).

Translocation of double-stranded DNA
Synthetic nanopores, in particular nanopores made in solid-state materials such as silicone nitride, can be fabricated with great accuracy (see "Nanopore Fabrication"). For the analysis of double stranded DNA , nanopores with diameters of 3-5 nanometer are ideal, due to two main reasons: First, the DNA enters the pore in a single file (unfolded) manner. Larger pores on the other hand permit folded and unfolded entries, thus complicating data analysis. Second, translocation time of the DNA in the small pores is substantially longer than the larger pores, facilitating the detection of short DNA molecules. Figure 3 displays characteristic DNA translocation events (800 bp) using a 3.5 nm pore (2M KCl).

References:

  1. Wanunu, M. and 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.
  2. 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.
  3. Meller, A., L. Nivon and D. Branton. (2001) Voltage-Driven DNA Translocations through a Nanopore. Phys. Rev. Lett. 86, 3435-3438.
  4. Wanunu, M., J. Sutin, B. Mcnally, A. Chowand A. Meller.(2008) DNA translocation governed by interactions with solid-state nanopores. Biophys. J. 95(10) 4716-25.























DNA translocation

Figure 1

Event Diagram poly(A) and poly(C)
Figure 2

dsDNA TL

Figure 3