Nucleobase Recognition by Truncated α-Hemolysin Pores

The α-hemolysin (αHL) protein nanopore has been investigated previously as a base detector for the strand sequencing of DNA and RNA. Recent findings have suggested that shorter pores might provide improved base discrimination. New work has also shown that truncated-barrel mutants (TBM) of αHL form functional pores in lipid bilayers. Therefore, we tested TBM pores for the ability to recognize bases in DNA strands immobilized within them. In the case of TBMΔ6, in which the barrel is shortened by ∼16 Å, one of the three recognition sites found in the wild-type pore, R1, was almost eliminated. With further mutagenesis (Met113 → Gly), R1 was completely removed, demonstrating that TBM pores can mediate sharpened recognition. Remarkably, a second mutant of TBMΔ6 (Met113 → Phe) was able to bind the positively charged β-cyclodextrin, am7βCD, unusually tightly, permitting the continuous recognition of individual nucleoside monophosphates, which would be required for exonuclease sequencing mediated by nanopore base identification.