Saturday, August 17, 2013

Nanopore Sequencing: Towards Reading and Writing?

Nanopore sequencing is an emerging technology that promises fast, easy and affordable way to 'read' the bases in DNA. While researchers are seeking the $1000 genome, nanopore sequencing (once refined) may be able to deliver under budget and on a time scale of minutes, not hours or days.

Other bloggers and science writers (at BiteSize Bio, among others) have done a great job covering this technology (several of which I complied at the end of this article in a short 'webibliography', or bibliography of websites). Here, I would like to speculate on the use of a nanopore for the synthesis (or writing) of a DNA sequence. 

FInding a cheaper and faster way to synthesize a DNA sequence is a big challenge, and one that with a growing urgency. I've previously highlighted the importance of meeting this challenge and some current attempts at solutions. The ideal solution may currently be residing in the realm of science fiction. As I mentioned in the previous article, solutions that employ a controllable polymerase have great potential. A recent article from the Akeson laboratory (Olasagasti, 2012; PMC3711841) shows that this may be possible. Indeed, Akeson and colleages are able to electronically control both the threading of DNA through a nanopore, as well as the synthesis of the threaded DNA. 

Select 'Read More' to see the rest of the article. What are your thoughts on nanopore sequencing? Do you think that it is feasible that this technology can be adapted in some way for a next-generation DNA sequencing solution?

Not lightning in a bottle, but ions through a pore

When boiled down to its essence, nanopore sequencing is quite simple. The technology relies upon measuring a small current passing from one side of a membrane to another. This membrane, placed within an electric field, has a small opening (the nanopore) through which ions that carry the current flow. Superficially similar phenomenon are exploited by bacteria and humans (and everything in between). Our nervous system, for example, sets up electrical fields across cell membranes by pumping ions out or drawing ions in. The gatekeepers (or nanopores) in the case of nerve cells are proteins that themselves respond to electrical potentials, helping to propagate an electric signal throughout the body.

Blocking or filling the nanopore will prevent the flow of ions, and cause a change in the measurable current of the system. If a DNA molecule is threaded through the nanopore, a change in the current will be observed that is characteristic of particular base passing through. This way, by measuring the varying changes in the electric field, it is possible to sequence a DNA molecule. Simply put, the nanopore system enables the DNA sequence to be electronically read. At least, that is the theory. Technical difficulties, such as a high error rate, have held back nanopore sequencing technology to date, although solutions are being actively sought.

Control of DNA nanopore sliding: It's electric

The laboratory of Dr. Mark Akeson (among others) at the Center for Biomolecular Science and Engineering has been pioneering DNA nanopore technology. In recent studies (such as Olasagasti et al, 2012 and Wilson et al, 2009), the Akeson lab would thread a DNA molecule through a alpha-hemolysin pore. The pore is such that only single strand of DNA can pass at one time. Once threaded through, a double stranded hairpin on one end (possibly both, but not necessarily) of the molecule would prevent it from sliding out (or translocating) in that direction. As such, the DNA molecule would be stuck, threaded through the pore but unable to fully translocate, like a screw passing through a piece of wood but with washers and nuts keeping it held in place (and unable to fall out completely).

When the DNA blocks the pore, it not only changes the ability of current to flow through the pore, but the movement of the DNA itself can be influenced by the electric field. Due to the placement of the molecular 'washer' (the hairpin), the DNA molecule can partially slide back and forward within the pore. The double stranded / single stranded junction is extended further by hybridizing an oligo to the remaining single strand portion on the same side of the pore (the purpose of this blocking oligo will become clear later). By applying the correct electrical field, the researchers would move the single stranded / double stranded DNA junction from the mouth (vestibule) of the pore into the solution. Once this junction is away from the pore and in the solution, it can be bound by a DNA polymerase (an enzyme that can synthesis a duplicate copy of DNA). The researchers call this procedure 'fishing', where the bait is a double stranded portion of DNA and the fish is the DNA polymerase (DNAP). The binding of DNAP can not be detected the same way a fisherman can feel the bite of a fish on their line. Instead, the line is reeled in during a step dubbed 'probing': the voltage is changed so as to draw the DNA back through the pore. Once retracted, the bound DNAP-DNA complex will alter the current flow through the pore. Because of this, capture of DNAP during the 'fishing' step can be detected through 'probing'. 

The system is somewhat more complex than what is described above, for technical reasons. The blocking oligomer is not necessary for creating the single stranded / double stranded junction, which is the point where new DNA synthesis would occur (this junction is formed by the hairpin structure). Instead, the junction formed by the blocking oligomer cannot support new synthesis (the 3' end of the oligomer does not hybridize properly). Instead of premature activity, the DNA synthesis is controlled in a precise manner through electronic removal of the blocking oligomer. Applying the proper force to draw the oligo-DNA junction into the pore vestibule weakens the binding of the oligo, and can lead to complete disassociation (without affecting the hairpin structure). 

Removal of the blocking oligomer frees the junction formed by the hairpin so that it may serve as a starting point for DNA synthesis. This synthesis extends the junction. The positioning of this junction can be voltage controlled with such precision that DNA synthesis can be controlled at one nucleotide base resolution. 

Nanopore synthesis: more than a pipe dream?

Clearly, the work by the Akeson group and others demonstrate that synthesis can be coupled to the movement of DNA through a nanopore. The resolution and control demonstrated in Olasagasti et al is particularly impressive. However, in these and similar studies, the synthesis is being controlled is that of DNAP replicating an existing sequence. While this is a great approach for studying DNAP dynamics and improving sequencing technologies, it is not a direct attempt at de novo DNA synthesis at a pore. The control developed here must be exploited in another way in order to serve as a replacement to the solid-state or microchip, stepwise DNA synthesis chemistry currently employed. Perhaps use of a DNAP capable of de novo synthesis, in absence of a template, can be employed in some fashion (Ramadan, et al 2004). Whatever the solution, a replacement technology will be badly needed for the ambitious field of synthetic biology. Realizing the full promises of nanopore sequencing may indeed foreshadow an equally elegant and revolutionary nanopore synthesis technology.

Selected Webibliography

Oxford Nanopore: One of the companies currently developing nanopore sequencing technology has a very informative (and visual pleasing) animation that describes the science behind the technology. A great place to start, if only to get a mental picture of what is occuring.

Another Revolution in NGS? Exciting Times Ahead with Nanopore Sequencing Technology: A BiteSizeBio article on nanopore sequencing, living up to the excellent standard of quality often demonstrated by that site. A concise and informative summary of the need for, and problems with, nanopore technology.  

DNA Sequencing, without the Fuss: Another great introductory article written for a general audience, but this time from Science magazine. Includes a diagram and user comments.

Wikipedia Entry: As it does with so many topics, Wikipedia provides a concise introduction to nanopore sequencing. Further reading (including the other links) is encouraged. The wikipedia page does a good job, however of surveying different variations on nanopore sequencing technology as well as companies attempting to commercialize these innovations.

Harvard Nanopore Group: The homepage for a collaborative nanopore research group at Harvard. While not as good of an introduction as Oxford nanopore or Wikipedia, research programs are described and links to the latest publications from the group are provided.

Disruptive nanpores: A short article from Nature that highlights the potential of nanopore sequencing, listing it as a 'method to watch'.

Nanopore article at NanoAll blog: Self-descriptive, a short introduction to nanopore sequencing technology at an interest nanotechnology blog. Apparently written for non-experts (i.e. not biologists)

References (Pub Med Central)

Henrik Stranneheim and Joakim Lundeberg (2012) Stepping stones in DNA sequencing  Biotechnology J. Vol 7 (9) 1063-1073 PMCID: PMC3472021

Olasagasti, F., (2012) Replication of Individual DNA Molecules under Electronic Control Using a Protein Nanopore. Nature Nanotechnology Vol 5 (11) pp. 798-806 PMCID: PMC3711841

Ramadan, K. (2004) De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase. Journal of Molecular Biology Vol 339 (2) pp. 395-404 PMID:15136041

Wlison, N., (2009) Electronic control of DNA polymerase binding and unbinding to single DNA molecules. ACS Nano Vol 3 (4) pp. 995-1003 PMCID: PMC2708927

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