We studied the unzipping of single molecules of double-stranded DNA by pulling one of their two strands through a narrow protein pore. Polymerase chain reaction analysis yielded the first direct proof of DNA unzipping in such a system. The time to unzip each molecule was inferred from the ionic current signature of DNA traversal. The distribution of times to unzip under various experimental conditions fit a simple kinetic model. Using this model, we estimated the enthalpy barriers to unzipping and the effective charge of a nucleotide in the pore, which was considerably smaller than previously assumed.

Broad-spectrum analysis of DNA and RNA samples is of increasing importance in the growing field of biotechnology. We show that nanopore measurements may be used to assess the purity, phosphorylation state, and chemical integrity of nucleic acid preparations. In contrast with gel electrophoresis and mass spectrometry, an unprecedented dynamic range of DNA sizes and concentrations can be evaluated in a single data acquisition process that spans minutes. Because the molecule information is quantized and digitally recorded with single-molecule resolution, the sensitivity of the system can be adjusted in real time to detect trace amounts of a particular DNA species.T he purity of a nucleic acid preparation and the chemical integrity of nucleic acid bases affect the efficiency of hybridization procedures, enzymatic reactions, and chemical modifications. These processes dictate the accuracy and reliability of routine biochemical and clinical investigations as well as the expanding field of array technologies. Although traditional techniques of electrophoresis, chromatography, and mass spectrometry can assess DNA or RNA sample purity and chemical integrity, the sensitivity of these methods is limited by the relative size and quantity of contaminating nucleic acids. More importantly, the resolution of these methods decreases with increasing DNA or RNA length. Sample evaluation is difficult for nucleic acids with Ͼ100 nt and is virtually impossible for those with Ͼ1,000 nt. In a process we call single-molecule electrophoresis, we show that a transmembrane nanopore can evaluate polynucleotides with Ͻ100 bases or Ͼ1,000 bases. The ensemble pattern produced by the individual interaction between the polymer and the nanopore reveals DNA sample properties in a manner unparalleled by other detection systems.The nanopore was formed by self-assembly of ␣-hemolysin (1), a robust channel-forming protein that has been used and engineered for stochastic sensing, characterization of small molecules, and detection and discrimination of individual DNA strands (2-7). A single ␣-hemolysin channel was embedded in a lipid membrane that partitioned a conducting solution into two chambers. A voltage bias across the membrane produced an ion current through the ␣-hemolysin and also drove negatively charged single-stranded DNA from the cis to the trans side of the channel. Translocations occurred on the microsecond timescale and were signaled by the partial blockage of ion current to 10-25% of the open pore current value (Fig.

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