“We believe that the D.N.A. is a code,” Francis Crick wrote to his twelve year old son in 1953. “That is, the order of the bases (the letters) makes one gene different to another.” Crick’s letter – bristling with excitement - contained one of the first descriptions of how DNA codes genetic information, and was shortly followed up with a paper published in Nature. With their comparable sizes and unique properties, nanostructures are now being exploited by researchers as tools to unpick the “code” hidden in the DNA of living organisms.

“Nanopores are being hailed as a potential next-generation DNA sequencer that could provide cheap, high-throughput DNA analysis,” suggest Spencer Carson and Meni Wanunu in a review published in Nanotechnology. The researchers, based at Northeastern University in the US, provide a detailed overview of the most significant advances in the field, which include both detection schemes and techniques for DNA motion control.

The review features in a focus collection the journal Nanotechnology has been preparing this year - with the help of guest editors Stuart Lindsay (Arizona State University, US) and Daniel Branton (Harvard University, US). The aim of the collection is to highlight the latest results and developments that use nanostructures to push the frontiers in DNA sequencing technology.

Nanopore devices traditionally operate by detecting an ion current through a nanopore as a molecule moves through it. The ionic current changes depending on the molecule or base passing through the pore at the time, which allows the base sequence of DNA molecules to be determined.

Lindsay together with colleagues at Arizona State University and Stony Brook University highlight the potential of an alternative approach described as “recognition tunnelling”. They monitor the tunnelling current between functionalised electrodes as the molecule passes between. Recognition tunnelling already shows promise, as it is sensitive to single bases and a working prototype has been demonstrated. In their paper Lindsay and his colleagues address some of the fundamental issues in the theory of recognition tunnelling, including the magnitude of the observed currents, fluctuations in currents and the frequency characteristics of these fluctuations.

New materials

The nanopores themselves can be produced in a range of materials, each with different advantages. In their contribution, Cynthia J Burrows, Henry S White and colleagues at Utah University in the US describe the potential for using nanopores in haemolysin – lipid and protein materials that occur naturally in living organisms. Their investigations look at using nanopores for identifying epigenetic markers, as well as damage to DNA resulting from oxidation or deamination, photochemical damage, and base release. They show that haemolysin pores can be successfully used to identify the cyclic complex BPDE when it is bound to the DNA chain. Metabolism of a carcinogenic precursor produces BPDE, so the technique may help determine an individual’s susceptibility to certain cancers.

The collection also includes work demonstrating the potential of new inorganic materials. In their theoretical work Ralph H Scheicher and Rodrigo G Amorim at Uppsala University in Sweden investigate the potential of silicene for nanopore sequencing applications. “Our findings suggest that silicene could be utilized as an integrated-circuit biosensor as part of a lab-on-a-chip device for DNA sequencing,” they conclude.

The verdict for another 2D material may be less positive. Cees Dekker and colleagues at the Delft University of Technology in the Netherlands report studies of the low-frequency noise in the ionic currents monitored as DNA translocates through nanopores. They compare graphene, which is attracting a great deal of attention for nanopore sequencing at present, with the more traditional material silicon nitride, and find that the noise is typically two orders of magnitude greater for graphene. “This is a drawback as it significantly lowers the signal-to-noise ratio in DNA translocation experiments,” they point out.

Scaling up

Silicon membranes are the focus for Jan Linnros and colleagues at the KTH Royal Institute of Technology in Sweden. They have assessed the feasibility and applications of electrochemically etched nanopore arrays as opposed to single pores. Existing fabrication techniques for inorganic nanopores, such as focused electron beam or ion beam drilling, do not readily scale up efficiently for fabricating arrays. However, Linnros and his team demonstrate the capability of electrochemically etching arrays of nanopores as small as 7nm in diameter through membranes 50-100nm thick. The KTH researchers also show that by labelling the molecules with fluorophores, optical detection techniques can be used to monitor the whole array in parallel.

With the mounting interest in the possible use of laser optics for manipulating DNA, and plasmonic techniques for measuring the translocation, the effects of associated temperature rises become increasingly relevant. Daniel V. Verschueren, Magnus P. Jonsson, and Cees Dekker at Delft University of Technology present the first systematic study of the temperature dependence of DNA translocation of DNA translocation through inorganic solid-state nanopores. They find that viscous drag on the untranslocated part of the DNA coil dominates the temperature dependence of translocation times, while the rate of molecules hitting pores is well described by a balance between diffusion and electrophoretic motion. The work provides a useful extension to existing theoretical models of these systems.

Wising up for new technologies

There is as yet still no consensus of opinion as to what degree the work of Watson and Crick was an extension of the work of Rosamund Franklin, who was also working on DNA in the 1950s although in a separate lab. Her work with Raymond Gosling containing “photograph 51”, an x-ray diffraction image that is likely to have contributed to Watson and Crick’s proposal of a double helical structure for DNA, was published as supporting evidence in the same issue of Nature as Crick and Watson’s paper. Gosling passed away on the 18 May earlier this year, but the controversy over the due accreditation for the discovery continues.

Altogether the combined efforts in DNA research, both in the 1950s and since, have invited science to engage in unravelling some of the most profound miracles of everyday life. “Science and everyday life cannot and should not be separated,” wrote the undergraduate Franklin to her father in defence of her scientific outlook, and her words proved very fitting for her later work. The potential applications of DNA sequencing and related technologies may make energetic demands on both the imaginations and judgement of the people wielding these new technologies. At the same time they have also inspired remarkable feats of scientific progress as you can find out in the articles of the Nanotechnology focus collection dedicated to DNA sequencing.