Mapping the Sensing Zone of Alpha-Hemolysin Using Immobilized DNA Containing a Single Abasic Residue

As efforts to sequence individual strands of DNA using biological nanopores continue to evolve, it has become increasingly important to understand the nature of the DNA-nanopore interaction. More specifically, characterizing the “sensing” zone inside the nanopore is an important part of understanding how nucleotide differentiation occurs. In order to characterize the sensing zone of the biological nanopore alpha-Hemolysin (αHL), we have examined how an adenine homopolymer containing a single abasic site blocks current flow through an αHL pore as the position of the abasic site is moved sequentially through the pore. In these experiments, single stranded DNA (ssDNA) oligonucleotides with a biotin (BTN) group at the 3’ terminus were allowed to complex with streptavidin. Due to the inability of the large streptavidin to translocate through the αHL, the attached oligonucleotide could be electrophoretically immobilized inside the pore. Creating and capturing numerous oligonucleotides in which the single abasic site was positioned progressively farther from the BTN termination (e. g. 7 adenines from the BTN end, 8 adenines from the BTN end, etc.) allowed a blocking current map of the αHL lumen to be developed. This blocking current map of αHL yielded a single peak, which is approximately two nucleotides in width, centered midway through the αHL pore where the beta-barrel meets the vestibule. This single peak corresponds to the primary sensing zone of αHL. Further experiments have demonstrated that both the width and location of the primary sensing zone can be altered by creating mutant αHL proteins in which the residues inside the αHL pore have been changed. These studies should prove integral in creating a class of mutant αHL pores capable of discriminating between single nucleotides in ssDNA.