Individual DNA and RNA molecules cause ionic current blockade signatures when driven through an alpha-hemolysin channel by an applied potential. This discovery suggests that a nano-scale pore could in principle provide direct, high-speed sequencing of single DNA or RNA molecules. Early investigations demonstrated that the single-stranded polymer traversal rate of 1--3 nucleotides per microsecond was too fast to resolve nucleotide sequences using standard patch clamp equipment. In order to increase resolution, we investigated methods to lengthen the DNA interaction with the pore. We first tested whether base pairing in a single strand of DNA would slow translocation. We found that a hairpin structure with at least three base pairs inserted in the middle of a forty-nucleotide Poly-dC strand measurably slowed the translocation time compared to a single linear stand with the same number of nucleotides. Single strands of DNA containing 2- to 10-base-pair hairpin structures increased blockade duration as a function of the number of base pairs in the hairpin stem; differences as small as two base pairs could be distinguished. Resolution of single base pair and single nucleotide differences was achieved when we examined blunt-ended hairpins, in which duplex stem length, base-pair mismatches, and loop length could be resolved on a millisecond time-scale. The alpha-hemolysin nanopore was particularly sensitive to differences at the terminus of a 9bp hairpin stein. Individual blockade signatures allowed discrimination of Watson-Crick base pairs and mismatches at the terminal position. Thermodynamic analysis of molecular stability accounted in large part for this sensitivity to small differences in structure. Several mechanisms are explored to explain the complex interaction between DNA, ionic current and the alpha-hemolysin pore.
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