The structural properties of the hemolysin nanopore shown in Figure 1 are relevant to understanding the mechanism of ionic current blockades. Here we will assume that the major component of the blockade occurs when a nucleic acid occupies the 5 nm long stem of the channel, and neglect contributions by the larger vestibule. The pore volume within the stem is 18 nm3, and the average diameter is 2.0 nm, with a limiting aperture of 1.5 nm at the neck of the pore.
Three parameters provide information about the nature of the linear polymer passing through a nanopore. The first is blockade amplitude, which is best normalized and expressed as a fraction or percentage of the open channel current, I/Io, where I is the blockade current and Io is the open channel current. I/Io has a characteristic value for many homopolymers of RNA and DNA, suggesting that it will be an important analytical feature of nanopore technology.
Although several factors could conceivably contribute to blockades of ionic current, the simplest to test experimentally is that the fractional volume of a linear nucleic acid strand occupying a pore will reduce the number of ions available to carry current. The contribution of fractional volume of the molecule occupying the pore stem has been tested by investigating blockades produced by several polyanions that vary in molecular volume yet have approximately the same charge density of phosphate along the strand. These blockade amplitudes were then compared with the fractional volume of the pore stem occupied by the polymer, taking into account not only the molecular volume of the polymer but also water of hydration on the polymer and pore walls. The exact conformation of a single strand of DNA in the channel is unknown, but for the purposes of this calculation a conservative estimate is 0.34 nm per base, the repeat distance of bases in a double helix. About 15 nucleotides would then be in the pore at any given time during translocation. (The pore is defined here as the portion of the hemolysin channel that penetrates the lipid bilayer and does not include the larger volume of the vestibule and channel mouth.) Assuming that each monomer of the nucleic acid has four waters of hydration (60 total), and that a single layer of water is bound to the interior surface of the pore (320 total), approximately 70% of the available pore volume would be occupied by a single strand of oligo(dA).
Similar calculations were made for oligo(dC), for an abasic strand of nucleic acid, and for polyphosphate, both of which also produce measurable ionic current blockades . The experimental values are in approximate agreement with the calculated values. From the results of this simple experiment, it can be concluded that blockade amplitude is largely a function of the fractional volume occupied by a linear polymer traversing a nanopore. The difference in total volume between purine and pyrimidine deoxyoligonucleotides in the a-hemolysin pore is only 0.3 nm3, which represents a 6% difference in the volume occupied by the molecules in the pore after correcting for water of hydration. This difference is just barely detectable as an average signal over noise and is produced by multiple nucleotides occupying the length of the pore. It follows that an improved pore having a smaller limiting aperture than that of the a-hemolysin channel will probably be required for single nucleotide resolution in nanopore sequencing applications.
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