Discrimination of single base substitutions in a DNA strand immobilized in a biological nanopore

Nanopores have been explored as highly sensitive sensors for detection and rapid sequencing of single molecules of DNA. To sequence DNA with a nanopore requires that adenine (A), cytosine (C), thymine (T), and guanine (G) produce distinct current signals as they traverse the pore. Recently, we demonstrated that homopolymers of adenine, cytosine, and thymine immobilized in the nanopore protein alpha-hemolysin (alphaHL) produced distinct current blockades dependent on their chemical orientation. To probe the detection limit of alphaHL, we examined immobilized single strands of T(40) DNA (polyT) with single base substitutions of A, C, and G at 12 positions on the strand occupying the stem region of alphaHL. We find blockade currents sensitive to base identity over most of these positions with the most sensitive region near the pore constriction. Adenine substitutions increase the measured blockade current to values intermediate to the polyT and polyA currents at a number of positions, while C substitutions increase the current to a level intermediate to polyT and polyC values in some positions, but decrease it below polyT in others. These changes in blockade current were also observed for G substitutions. These results indicate that total blockade currents measured in alphaHL arise from nucleotides at multiple locations and thus are not uniquely attributable to an individual base in a specific position, a finding consistent with a recently published study. The measurements of C and G substitutions also suggest that blockade current may be modulated through interactions between nucleotides and the pore interior at multiple sites in alphaHL.