Nanopore technology for DNA sequencing is constantly being refined and improved. In strand sequencing a single strand of DNA is fed through a nanopore and subsequent fluctuations in the current are measured. A major hurdle is that the DNA is translocated through the pore at a rate that is too fast for the current measurement systems. An alternative approach is "exonuclease sequencing", in which an exonuclease is attached to the nanopore that is able to process the strand, cleaving off one base at a time. The bases then flow through the nanopore and the current is measured. This method has the advantage of potentially solving the translocation rate problem, as the speed is controlled by the exonuclease. Here we consider the practical details of exonuclease attachment to the protein alpha hemolysin. We employ molecular dynamics simulations to determine the ideal (a) distance from alpha-hemolysin, and (b) the orientation of the monophosphate nucleotides upon release from the exonuclease such that they will enter the protein. Our results indicate an almost linear decrease in the probability of entry into the protein with increasing distance of nucleotide release. The nucleotide orientation is less significant for entry into the protein.
Keywords: alpha-hemolysin; exonuclease sequencing; molecular dynamics; nanopore sequencing.