Nanopore unstacking of single-stranded DNA helices

The force-induced unstacking of individual single-stranded (ss) homopolymeric DNA helices during translocation through an αhemolysin protein nanopore by electrophoresis was demonstrated. Single-stranded homopolymeric nucleic acids had been shown to assume a rigid secondary helical structure that is stabilized by aromatic base stacking between consecutive bases along the sugar-phosphate backbone. DNA translocation through α-hemolysin gave rise to a transient reduction in ionic current. The kinetic steps in the ssDNA translocation involved cooperative unstacking of two nucleotides in the poly(dC) string and five nucleotides in the (polydA) string. The cooperative unstacking energy of a ssDNA homopolymer deduced from its translocation kinetics depended on its nucleotide sequence and orientation. Results show that the energy barrier of unstacking depends on the sequence and orientation of the ssDNA.