Oxford Nanopore Technologies

DNA Sequencing (exonuclease)

Exonuclease sequencing: Combining a protein nanopore and processive enzyme for the sequential identification of DNA bases as they pass through the pore.

Alpha haemolysin nanopore showing cyclodextrin adapter molecule (the DNA binding site).

"Wild type" (naturally occurring) α-hemolysin nanopore alone is not capable of DNA sequencing. Oxford Nanopore is using protein engineering techniques to adapt the nanopore for the detection of DNA bases.

A key achievement in adapting the nanopore has been the covalent attachment of a cyclodextrin molecule to the inside surface of the nanopore, This acts as a binding site for individual DNA bases and allows accurate measurement of their passage through the nanopore binding site.

Oxford Nanopore is addressing the issue of how DNA is passed through the nanopore by adopting a novel exonuclease sequencing approach. This takes advantage of the ability of exonuclease enzymes to process DNA and cleave individual bases from the end of a DNA strand. The exonuclease is attached to the nanopore in order to deliver individual DNA bases in sequence into the nanopore for rapid and accurate identification.

Other methods of nanopore sequencing
Oxford Nanopore is also working on other methods of using nanopores to sequence DNA.

'Strand sequencing': analysing intact strands of DNA rather than cleaving individual bases
'hybrid pores': embedding protein nanopores into synthetic membranes rather than lipid bilayers
'solid state' nanopores as holes made directly in man made materials ("solid state" nanopores)

Oxford Nanopore's network of collaborators continue to work on these developments and the company has leading intellectual property in these areas (see press releases: 1, 2)

The DNA sequencing system
Oxford Nanopore's sensing system is modular. Our proprietary array chip is combined with electronics and other hardware for signal capture. The array chip can then be populated with a range of adapted nanopores. These may be tailored for the analysis of DNA, proteins or other analytes.

The system for exonuclease sequencing is shown below:

Nanopore	Array Chip	Hardware
A protein nanopore, alpha-hemolysin An exonuclease is coupled with the nanopore. This enzyme is responsible for capturing DNA strands, sequentially cleaving individual bases from the strand, and directing the bases into the aperture of the nanopore. An engineered cyclodextrin sensor is covalently attached to the inside surface of the nanopore. This acts as a binding site for DNA bases as they pass through the pore. click here for more information on nanopore chemistry.	A lipid bilayer is created over a microwell that contains a pair of electrodes on either side of the bilayer. Adapted alpha-hemolysin nanopores are introduced into the bilayer, creating a single hole. The lipid bilayer has a high electrical resistance and so when an electrical potential is applied across this membrane, a current flows only through the nanopore, carried by the ions in salt solutions that bathe both sides of the bilayer. DNA sample is introduced into the top layer. As the exonuclease directs individual DNA bases, in sequence, through the nanopore, each base transiently binds at the binding site. During the binding event, the current through the nanopore is disturbed, creating a characteristic signal for each type of base. The signal for each base can be easily distinguished. The electrical current trace provides a record of the sequence of bases passing through the nanopore. Click here for more information about our array chip.	To achieve high-throughput sequencing, this system is run in parallel in an array chip. By combining the traces from each well in the array chip, data reassembly can be performed and the genome sequence will be constructed. Simple instrumentation is required to operate the array chip, and record the resulting electrical signals. Direct electrical detection and potential long read lengths promises simpler bioinformatics click here for more information about nanopore informatics

Nanopore

Array Chip

Hardware

A protein nanopore, alpha-hemolysin
An exonuclease is coupled with the nanopore. This enzyme is responsible for capturing DNA strands, sequentially cleaving individual bases from the strand, and directing the bases into the aperture of the nanopore.
An engineered cyclodextrin sensor is covalently attached to the inside surface of the nanopore. This acts as a binding site for DNA bases as they pass through the pore.
click here for more information on nanopore chemistry.

A lipid bilayer is created over a microwell that contains a pair of electrodes on either side of the bilayer.
Adapted alpha-hemolysin nanopores are introduced into the bilayer, creating a single hole.
The lipid bilayer has a high electrical resistance and so when an electrical potential is applied across this membrane, a current flows only through the nanopore, carried by the ions in salt solutions that bathe both sides of the bilayer.
DNA sample is introduced into the top layer. As the exonuclease directs individual DNA bases, in sequence, through the nanopore, each base transiently binds at the binding site.
During the binding event, the current through the nanopore is disturbed, creating a characteristic signal for each type of base. The signal for each base can be easily distinguished.
The electrical current trace provides a record of the sequence of bases passing through the nanopore.
Click here for more information about our array chip.

To achieve high-throughput sequencing, this system is run in parallel in an array chip.
By combining the traces from each well in the array chip, data reassembly can be performed and the genome sequence will be constructed.
Simple instrumentation is required to operate the array chip, and record the resulting electrical signals.
Direct electrical detection and potential long read lengths promises simpler bioinformatics
click here for more information about nanopore informatics