Nucleobase recognition in ssDNA at the central constriction of the alpha-hemolysin pore

Nanopores are under investigation for single-molecule DNA sequencing. The alpha-hemolysin (alphaHL) protein nanopore contains three recognition points capable of nucleobase discrimination in individual immobilized ssDNA molecules. We have modified the recognition point R(1) by extensive mutagenesis of residue 113. Amino acids that provide an energy barrier to ion flow (e.g., bulky or hydrophobic residues) strengthen base identification, while amino acids that lower the barrier weaken it. Amino acids with related side chains produce similar patterns of nucleobase recognition providing a rationale for the redesign of recognition points.