Nanopore sequencing

Nanopore sequencing is a method under development since 1995 ^[1][2] for determining the order in which nucleotides occur on a strand of DNA.

A nanopore is simply a small hole, of the order of 1 nanometer in internal diameter. Certain transmembrane cellular proteins act as nanopores, and nanopores have also been made by etching a somewhat larger hole (several tens of nanometers) in a piece of silicon, and then gradually filling it in using ion-beam sculpting methods which results in a much smaller diameter hole: the nanopore.

alpha-hemolysin pore (made up of 7 identical subunits in 7 colors) and 12-mer single-stranded DNA (in white) on the same scale to illustrate DNA effects on conductance when moving through a nanopore. Below is an orthogonal view of the same molecules.

The theory behind nanopore sequencing has to do with what occurs when the nanopore is immersed in a conducting fluid and a potential (voltage) is applied across it: under these conditions a slight electric current due to conduction of ions through the nanopore can be observed, and the amount of current is very sensitive to the size and shape of the nanopore. If single nucleotides (bases) or strands of DNA pass through the nanopore, this can create a characteristic change in the magnitude of the current through the nanopore.

DNA could be passed through the nanopore for various reasons. For example, electrophoresis might attract the DNA towards the nanopore, and it might eventually pass through it. Or, enzymes attached to the nanopore might guide DNA towards the nanopore. The scale of the nanopore means that the DNA may be forced through the hole as a long string, one base at a time, rather like thread through the eye of a needle. As it does so, each nucleotide on the DNA molecule may obstruct the nanopore to a different, characteristic degree. The amount of current which can pass through the nanopore at any given moment therefore varies depending on whether the nanopore is blocked by an A, a C, a G or a T. The change in the current through the nanopore as the DNA molecule passes through the nanopore represents a direct reading of the DNA sequence. Alternatively, a nanopore might be used to identify individual DNA bases as they pass through the nanopore in the correct order - this approach has been shown by Oxford Nanopore Technologies and Professor Hagan Bayley.^[3]

The potential is that a single molecule of DNA can be sequenced directly using a nanopore, without the need for an intervening PCR amplification step or a chemical labelling step or the need for optical instrumentation to identify the chemical label. As of July 2010, information available to the public indicates that nanopore sequencing is still in the development stage, with some laboratory-based data to back up the different components of the sequencing method, but not yet commercially available, parallelized, routineized, nor cost-effective enough yet to compete with out "next generation sequencing" methods. Nanopore-based DNA analysis techniques are being industrially developed by Oxford Nanopore Technologies (developing direct exonuclease sequencing and strand sequencing using protein nanopores, and solid-state sequencing through internal R&D and collaborations with academic institutions), NabSys (using a library of DNA probes and using nanopores to detect where these probes have hybridized to single stranded DNA) and NobleGen(using nanopores in combination with fluorescent labels). IBM has noted research projects on computer simulations of translocation of a DNA strand through a solid-state nanopore, but not projects on identifying the DNA bases on that strand.

One challenge for the 'strand sequencing' method is in refining the method to improve its resolution to be able to detect single bases. In the early papers methods, a nucleotide needed to be repeated in a sequence about 100 times successively in order to produce a measurable characteristic change. This low resolution is because the DNA strand moves rapidly at the rate of 1 to 5μs per base through the nanopore. This makes recording difficult and prone to background noise, failing in obtaining single-nucleotide resolution. The problem is being tackled by either improving the recording technology or by controlling the speed of DNA strand by various protein engineering strategies.^[4] More recently effects of single bases due to secondary structure or released mononucleotides have been shown.^[5][6] Professor Hagan Bayley, founder of Oxford Nanopore, recently proposed that creating two recognition sites within an alpha hemolysin pore may confer advantages in base recognition.^[7]

One challenge for the 'exonuclease approach',^[8] where a processive enzyme feeds individual bases, in the correct order, into the nanopore, is to integrate the exonuclease and the nanopore detection systems. In particular,^[9] the problem is that when an exonuclease hydrolyzes the phosphodieseter bonds between nucleotides in DNA, the subsequentially released nucleotide is not necessarily guaranteed to directly move in to, say, a nearby alpha-hemolysin nanopore. One idea^[9] is to attach the exonuclease to the nanopore, perhaps through biotinylation to the beta barrel hemolsyin. The central pore of the protein may be lined with charged residues arranged so that the positive and negative charges appear on opposite sides of the pore. However, this mechanism is primarily discriminatory and does not constitute a mechanism to guide nucleotides down some particular path.

Commercialization

Agilent Laboratories was the first to license and develop nanopores ^[10] but does not have any current disclosed research in the area.

The company Oxford Nanopore Technologies in 2008 licensed technology from Harvard, UCSC and other universities ^[11] and is developing protein and solid state nanopore technology with the aim of sequencing DNA and identifying biomarkers, drugs of abuse and a range of other molecules.

Sequenom licensed nanopore technology from Harvard in 2007^[12] using an approach that combines nanopores and fluorescent labels. This technology was subsequently licensed to Noblegen.^[13]

NABsys was spun out of Brown University and is researching nanopores as a method of identifying areas of single stranded DNA that have been hybridized with specific DNA probes.

References

1. ^ Church, G.M.; Deamer, D.W., Branton, D., Baldarelli, R., Kasianowicz, J. (1998). "US patent # 5,795,782 (filed March 1995) Characterization of individual polymer molecules based on monomer-interface interaction". http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&co1=AND&d=PTXT&s1=5,795,782.PN.&OS=PN/5,795,782&RS=PN/5,795,782. 
2. ^ Kasianowicz, JJ; Brandin E, Branton D, Deamer DW (1996-11-26). "Characterization of individual polynucleotide molecules using a membrane channel.". Proc Natl Acad Sci USA 93 (24): 13770–3. doi:10.1073/pnas.93.24.13770. PMC 19421. PMID 8943010. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=19421. 
3. ^ Clarke J, Wu HC, Jayasinghe L, Patel A, Reid A, Bayley H (2009). "Continuous base identification for single-molecule nanopore DNA sequencing". Nature Nanotechnology 4 (4): 265–270. doi:10.1038/nnano.2009.12. PMID 19350039. http://www.nature.com/nnano/journal/v4/n4/full/nnano.2009.12.html. 
4. ^ Hagan Bayley, Sequencing single molecules of DNA, Current Opinion in Chemical Biology, Volume 10, Issue 6, Model systems / Biopolymers, December 2006, Pages 628-637, ISSN 1367-5931, DOI: 10.1016/j.cbpa.2006.10.040. (http://www.sciencedirect.com/science/article/B6VRX-4MCWB5T-1/2/7090e8c122adfca35c1a2e99fa177f7c)
5. ^ Ashkenas, N; Sánchez-Quesada J, Bayley H, Ghadiri MR (2005-02-18). "Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores". Angew Chem Int Ed Engl 44 (9): 1401–4. doi:10.1002/anie.200462114. PMC 1828035. PMID 15666419. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1828035. 
6. ^ Winters-Hilt, S; Vercoutere W, DeGuzman VS, Deamer D, Akeson M, Haussler D (February 2003). "Highly accurate classification of Watson-Crick basepairs on termini of single DNA molecules". Biophys J. 84 (2 Pt 1): 967–76. doi:10.1016/S0006-3495(03)74913-3. PMC 1302674. PMID 12547778. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=1302674. 
7. ^ Stoddart D, Maglia G, Mikhailova E, Heron A, Bayley H (2010). "Multiple Base-Recognition Sites in a Biological Nanopore: Two Heads are Better than One". Angew Chem Int Ed Engl 49 (3): 556–9. doi:10.1002/anie.200905483. PMID 20014084. http://www3.interscience.wiley.com/journal/123210758/abstract?CRETRY=1&SRETRY=0. 
8. ^ Astier, Y; Braha O, Bayley H (2006-02-08). "Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter". J Am Chem Soc. 128 (5): 1705–10. doi:10.1021/ja057123. PMID 16448145. 
9. ^ ^{a b} Rusk, Nicole (2009-04-01). "Cheap Third-Generation Sequencing". Nature Methods 6 (4): 244–245. doi:10.1038/nmeth0409-244a. 
10. ^ "Agilent Laboratories, Harvard University Collaborate On Development of Breakthrough Technology for the Analysis of Nucleic Acids". Business Wire. 2001-05-21. http://findarticles.com/p/articles/mi_m0EIN/is_2001_May_21/ai_74803301. 
11. ^ "Harvard University And Oxford Nanopore Technologies Announce Licence Agreement To Advance Nanopore DNA Sequencing". http://www.medicalnewstoday.com/articles/117827.php. 
12. ^ "Sequenom to Develop Third-Generation Nanopore-Based Single Molecule Sequencing Technology". http://www.freshnews.com/news/biotech-biomedical/article_39927.html. 
13. ^ "Noblegen commercialise optical sequencing". http://www.genomeweb.com/sequencing/noblegen-commercialize-bus-optical-readout-nanopore-sequencing-tech. 

1 Nature NanotechnologyFebruary 2009, Volume 4 No 2 pp71–133

Reviews

• Zwolak M, Di Ventra M. Colloquium: Physical approaches to DNA sequencing and detection. Reviews of Modern Physics 80, 141 (2008)
• Astier Y, Braha O, Bayley H: Towards single molecule DNA sequencing. J. AM. CHEM. SOC. 2006, 128, 1705-1710 9 1705
• Fologea D el al. Detecting single stranded DNA with a solid state nanopore. Nano Lett. 2005 Oct;5(10):1905-9. PMID 16218707
• Deamer DW, Akeson M. Nanopores and nucleic acids: prospects for ultrarapid sequencing. Trends Biotechnol. 2000 Apr;18(4):147-51. PMID 10740260
• Church, George M. Genomes for all. Scientific American. 2006 Jan;294(1):52. PMID 16468433
• Xu M. S., Fujita D., Hanagata N. "Perspectives and challenges of emerging single-molecule DNA sequencing technologies". Small 2009, 5 (23), 2638-49.