Engineered nanopores made from proteins can be employed in a variety of biotechnology applications, including molecular sensing. They also make good DNA sequencers. One particular such nanopore, the alpha-hemolysin (αHL) appears to be especially well suited to detecting individual bases of DNA and RNA, with shorter pores being even better.

Last year, a team led by Hagan Bayley of the Department of Chemistry at the University of Oxford discovered that “truncated-barrel mutants” (TBMs) of αHL might also possibly be used as pores to sequence DNA bases. The researchers have now gone a step further in their new work by reducing the number of reading heads in the αHL and have so improved these protein pores as sequence readers.

Sequencing DNA bases with nanopores

DNA, which is often called the blueprint of life, is composed of four chemical bases: adenine (A), guanine (G), cytosine (C) and thymine (T), which are paired together in a complementary fashion (A to T and C to G) and ordered in a specific sequence. This sequence plays a vital role in biology since it stores information important for constructing the protein machinery that makes a living cell work.

Sequencing DNA bases with nanopores is a relatively new concept. The technique involves electrically detecting each base pair as the DNA molecule translocates through the pore by measuring either the voltage across the membrane or the current through the pore.

Shortened αHL barrel

Bayley and colleagues studied the 5-nm long “transmembrane β barrel” of the αHL pore. This membrane comprises the base recognition region of the protein, and the “wild-type” pore contains three broad recognition sites: R1, R2 and R3, all located at different regions. “Previous work has shown that the αHL pore can separate out all four DNA bases at one or more of these recognition sites,” explains team member Mariam Ayub.

The team has now found that a TBM (TBMΔ6) in which the αHL barrel has been shortened by around 16 angstroms is also able to successfully identify all four DNA bases, and without any breaks in current recording. This means that the structure may be able to sequence DNA just as well as more routinely employed DNA sequencing techniques that rely on complicated and expensive chemistry to “amplify” the DNA.

Reducing the number of recognition sites and scaling up

The shortened αHL pore is as efficient because the number of recognition sites in the pores has been reduced – from three to two – so making for sharper reading heads. The approach should generally be applicable to a variety of pore-forming proteins, says Ayub, and our work suggests that such radical protein engineering is a good way to improve these structures for identifying DNA bases.

An added bonus of the protein pore system is that it could be scaled up (or “parallelized”), which would make it competitive with highly parallel second generation sequencing technologies, she adds. “Indeed, an array of 106 nanopores each reading 100 bases per second could sequence a human genome cheaply and in around 10 minutes, a feat that could make genomic medicine readily available to all,” she tells

The research is detailed in ACS Nano DOI: 10.1021/nn5060317.

For more on DNA sequencing keep an eye out for articles in the Nanotechnology focus collection.