Figure 3. ssDNA and dsDNA blockades of ClyA-CS.

At +100 mV, the addition of 2.0 μM of a biotinylated ssDNA (5a) to the cis side of ClyA-CS nanopores in the presence of 0.6 μM neutravidin (tetramer) provoked transient current blockades (level 2*₊₁₀₀=1.24±0.02 nA, mean±s.d., I_RES+100=0.69±0.04, mean±s.d., n=3), indicating that ssDNA entered the lumen of the pore but only transiently. The subsequent addition of 2.0 μM of the complementary ssDNA strand (5b) to the cis compartment converted the current blockades into permanent level 1₊₁₀₀ blocks (1.22±0.13 nA, mean±s.d., I_RES+100=0.67±0.01, mean±s.d., n=3), indicating that the dsDNA so formed spanned the entire length of the pore. After each DNA capture, the open pore was regenerated by manual reversal of the potential to −100 mV. Spikes above and below the open pore current level represent capacitive transients. The electrical recordings were carried out in 2.5 M NaCl, 15 mM Tris HCl, pH 7.5, at 22 °C. Data were recorded by applying a 10-kHz low-pass Bessel filter and using a 20μs (50 kHz) sampling rate.