Oligomers of poly[U] caused transient blockades in the α-hemolysin single channel current. At the first arrow, a potential of −120 mV was applied across the membrane (cis side negative). This voltage caused a continuous current of −120 pA to flow. At the second arrow, poly[U] of mean length 210 bases was stirred into the cis compartment to a final concentration of 0.1 mg/ml. The polynucleotides caused short-lived current blockades. The Inset (expanded time scale) shows two typical blockades with lifetimes of 300 and 1300 μs. For the purposes of illustration, the low time resolution current recordings (a total of 4 sec is shown here) were digitally filtered using an exponential smoothing moving average algorithm.

The characteristic lifetimes of channel blockades caused by poly[U] (0.1 mg/ml; mean length, 210 nt) fell within three well-defined peaks. The mean lifetimes corresponding to the three peaks were determined by fitting the sum of three Gaussians to the data.

Poly[U]-induced channel blockade lifetimes were proportional to (a) mean polymer length and (b) inversely proportional to applied voltage. The plots show lifetimes for (a) peaks 1 (+), 2 (□), and 3 (•) in experiments using V = −120 mV with 13 different size selected poly[U]s and (b) for peaks 2 (□), and 3 (•) with poly[U] of mean length 215 nt at the indicated voltages. Although the peak 1 lifetime appeared to be independent of the applied voltage (data not shown), because this lifetime (≈100 μs) was barely a factor of 2 greater than the time resolution of our system, a slight voltage dependence could not be ruled out.

Single-stranded DNA traversed the α-hemolysin pore. The PCR-amplified product (lane 1) of a 40 μl sample of the trans chamber compared with (lane 2) the similarly amplified product of a 10⁹-fold diluted sample of the cis chamber. All samples were taken following an 8 channel experiment in which a mixture of 150-nt single-stranded DNA (see Materials and Methods) and 100-nt double-stranded DNA was added to the cis chamber. Material in the trans chamber sample reflects accumulation during application of −120 mV (cis chamber negative) for 42 min. Lanes 3-7: example of competitive PCR analysis (25) to show accumulation of single-stranded molecules in the trans chamber. The results show that the 40-μl sample used here contained ≈400 of the 150-nt single-stranded molecules. To each of five 40-μl samples of the trans chamber were mixed (lanes 3–7) 0, 100, 200, 400, or 800 molecules of a competing 120-nt DNA that was PCR amplified using the same primers as the 150-nt DNA. The position of the amplified 150-, 120-, and 100-nt polymers is shown at left. Lanes 1 and 3 show independently amplified 40-μl samples of the same trans chamber.

Enzymatic hydrolysis of poly[U] caused a transient increase in the rate of oligonucleotide-induced channel blockades. In two separate experiments, at time 0, ribonuclease A (bovine pancreatic RNase Type XII-A; Sigma or Worthington) was added to the cis chamber containing either 0.13 mg/ml poly[U] (•) (mean length, 1100 nt) or 0.27 mg/ml poly[A] (□) (mean length, 20,000 nt; Fluka). The final ribonuclease concentrations were 0.04 mg/ml (poly[U] experiment) or 0.13 mg/ml (poly[A] experiment). Blockade rates were calculated from single channel recordings. The Inset shows (null)/1;3-sec sample traces for the poly[U] experiment before (Upper) and ≈30 sec after (Lower) addition of ribonuclease. Similar experiments with a poly[U] sample of greater length (3000 nt) yielded results comparable to those obtained with the 1100-nt poly[U]. The current was digitized using a SONY/Dagan DAT recorder at 48,000 points/sec.