An analysis of mismatched duplex DNA unzipping through a bacterial nanopore

A 50-base Guide strand was synthesized that consisted of a central 10-base probe sequence flanked by two tracts of 20 adenine residues. Target sequences of 10 bases containing up to three mismatches were prepared and hybridized to the Guide strand in 1 M KCl. The transport of these constructs through single alpha-hemolysin pores was analysed by measuring the current blockade as a function of time. Complementary dsDNA takes significantly longer (840 +/- 60 mus) to pass through the pore than a sequence of the same length containing a single (590 +/- 45 mus) and a double (270 +/- 50 mus) mismatch. Constructs involving three mismatches were indistinguishable from Guide ssDNA transport (120 +/- 30 mus). The results suggest that dsDNA must unzip as it is transported through the nanopore. Duplexes containing mismatches unzip more quickly and can be distinguished from those with perfect complementarity.