Nanopore Chemistry

Oxford Nanopore is intially working with protein nanopores.  The heptameric protein alpha-hemolysin (AHL), in nature excreted by bacteria as a tool for forming pores in target membranes, it offers many advantages in the industrial setting.  AHL is easily and cheaply produced using bacteria, it can be modified with angstrom-level precision using biochemical or molecular biology techniques, and the protein is very robust.

The alpha-hemolysin nanopore has been characterised and studied at great length by the founder Professor Hagan Bayley, other Company collaborators, and extensively within the Company. 

AHL is a key component of Oxford Nanopore's DNA analysis methods (exonuclease sequencing and strand sequencing) and also for protein analysis and other applications.  In the future protein nanopore arrays may be replaced by solid-state arrays, manufactured holes in synthetic materials.  Oxford Nanopore has research and IP partnerships with Harvard in this area.

Exonuclease DNA sequencing
For exonuclease sequencing, an AHL nanopore is combined with a processive enzyme that cleaves individual bases from the end of a single strand of DNA.  Individual bases are introduced to the nanopore and are identified as they pass through.

In 2008, Oxford Nanopore published a paper in Nature Nanotechnology that demonstrated chemical modifications to the nanopore. These allow the identification of individual DNA bases to a standard commensurate with a high resolution DNA sequencing technology.  In addition the paper demonstrated the direct identification of the modified base Methylated Cytosine.  To order an e-print of this paper click here, choosing 'e-print' from the drop own box.

The following figure shows an electronic trace of  DNA bases in solution entering the nanopore. The bases individually, transiently bind to the cyclodextrin adapter. Each time a base passes through the pore there is a disruption in the current.  The diagram shows four different magnitudes of disruption which can be classified as C, G, A or T.








These data points can be displayed on a histogram, shown below, to illustrate how the identify of a single base is calculated.  As the signal is electronic rather than optical, if there is a base ambiguity it is two-way rather than four-way.
















The following histogram shows the addition of the modified base methylated cytosine, which can be directly distinguished from the four standard bases.  
















Strand DNA sequencing
Strand sequencing requires the identification of individual bases on a single strand as it passes through the nanopore. Oxford Nanopore is exploring this development method through internal R&D and external collaborations spanning research projects and IP licensing.

A key challenge for this approach is the successful discrimination of individual bases on the strand, as multiple bases occupy the aperture of the nanopore simultaneously. Progress has recently been made in the laboratories of Professor Hagan Bayley, click here for reference.

Also, the speed of the DNA strand as it translocates the nanopore must be controlled in order to achieve accurate base resolution.  Oxford Nanopore is exploring various methods of using molecular motors such as enzymes to translocate the DNA strand.

Other target analytes
Other modifications have been made to a protein nanopore to allow the identification of different target analytes, including proteins and small molecules.  For a full list of publications click here. Oxford Nanopore is currently exploring the use of other modifications to the nanopore in combination with its sensor chip technology.

Join our Chemistry team
Oxford Nanopore has a large team, led by Dr John Milton, working on the development of our nanopore chemistry, and we are seeking talented chemists to help us progress the technology still further.  Visit our careers page for more information.