Abstract
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.
Highlights
For the purposes of clarity when reporting our results, we use two terms for the behaviour of the nucleotide with respect to the protein: “capture” and “possible capture”. These are defined as follows: When the entire cytosine monophosphate (CMP) is below the ring of C-alpha atoms of N17, it is regarded as captured as over all simulations we observed no examples of exit from the vestibule entrance once interacting with the protein below this region
The final alternative is when simultaneously parts of the CMP are above and below the N17 ring, which we refer to as possible capture, as it was observed that CMP in this region, which we describe as the “edge” of the vestibule entrance, is capable of either entering the vestibule or diffusing into solution
Our simulations predict that optimisation of alpha-hemolysin for nanopore sequencing, which incorporates an exonuclease enzyme for cleaving nucleotides from a strand of DNA, must consider the protein-exonuclease distance of nucleotide release
Summary
DNA sequencing using nanopores is a method that allows for direct analysis of genomic DNA [1,2,3,4,5,6,7,8]. The pore forming toxin, alpha-hemolysin (αHL) from S. aureus [11] is perhaps the best studied example of a proteinaceous nanopore for DNA sequencing [12]. This protein has two domains, the vestibule or cap domain and the transmembrane pore domain (Figure 1) [11]. Further details of the methodology are provided in the Experimental Section
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