Research published in Nature Nanotechnology shows a new method of enzyme-controlled movement of a single strand of DNA through a protein nanopore. The paper, by researchers at the University of California Santa Cruz, represents a key step towards nanopore sequencing of DNA strands. Nanopore single-molecule analysis is a new generation of technology currently being developed for label-free, electronic DNA sequencing and protein analysis. This has potential performance and cost advantages over existing technologies.
The publication describes the observation of single stranded DNA (ssDNA) as it translocates through a protein nanopore, alpha hemolysin (AHL). Movement of the ssDNA was controlled by polymerase-facilitated replication of individual DNA molecules. This movement could be initiated under electronic control. Polymerase activity was shown to be blocked in solution when the ssDNA was not at the nanopore opening, however capture of the strand by the pore removes a blocking strand of nucleotides and allows the polymerase to function on the trapped strand.
UCSC researchers are collaborating with Oxford Nanopore Technologies Ltd in the development of a new generation of electronic, single-molecule DNA sequencing technology. In the 'strand sequencing' method, current through a nanopore is measured as a DNA polymer passes through that pore. Changes in this current are used to identify the DNA bases on the DNA molecule, in sequence. This paper addresses a key challenge for DNA strand sequencing: fine control of the translocation of the DNA strand through the nanopore, at a rate that is consistent and slow enough to enable accurate identification of individual DNA bases.
The Nature Nanotechnology work shows for the first time that the motion of a strand can be controlled using electronic feedback and that an enzyme can move a strand against a field while located on top of the nanopore.
"The techniques described in this paper are an advance towards electronic, single molecule DNA sequencing of DNA strands" said investigator Professor Mark Akeson of the University of California, Santa Cruz. "Electronic control of DNA translocation through a protein nanopore is a scientific goal that we have strived towards for years and these methods are now forming the basis for further work in our laboratories. We are excited by our collaboration with Oxford Nanopore, whose parallel nanopore sensing strategy is impressive."
Reference: Replication of individual DNA molecules under electronic control using a protein nanopore. Felix Olasagasti, Kate R. Lieberman, Seico Benner, Gerald M. Cherf, Joseph M. Dahl, David W. Deamer and Mark Akeson Nature Nanotechnology September 2010.
DOI: 10.1038/NNANO.2010.177, (subscription needed)
DOI: 10.1038/NNANO.2010.177, (subscription needed)
Work conducted in this paper
In this Nature Nanotechnology paper, DNA replication was catalyzed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF) in order to drive ssDNA through the nanopore. The T7DNAP enzyme advanced on a DNA template against an 80 mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond time scale for hundreds of individual DNA molecules in series. When using the KF enzyme, nucleotide additions were not observed when the enzyme was directly on the pore, but using electronic feedback, KF enzymes were allowed to act on the strand while in the solution above the pore, resulting in a controlled movement of the strand.
Base identification during strand sequencing
In addition to achieving fine control of DNA translocation through a nanopore, a key challenge for strand sequencing is accurate identification of individual nucleotides on ssDNA. When passing through AHL,10-15 bases on a ssDNA polymer will span the pore's central channel. Strategies are in development for distinguishing single bases, for example researchers at the University of Oxford have previously published a method to correctly identify individual nucleotides on ssDNA immobilised within an AHL nanopore. Further work continues at Oxford Nanopore and in the laboratories of the Company's collaborators.
Oxford Nanopore Technologies Ltd
Oxford Nanopore Technologies Ltd is developing a revolutionary technology for direct, electrical detection and analysis of single molecules. The platform is designed to offer substantial benefits in a variety of applications. The Company's lead application is DNA sequencing, but the platform is also adaptable for protein analysis for diagnostics and drug development and identification of a range of other molecules for security & defence and environmental monitoring. The technology is modular and highly scalable, driven by electronics rather than optics.
The Company's first generations of DNA sequencing technology, Exonuclease sequencing and Strand sequencing, combine a protein nanopore with a processive enzyme, multiplexed on a silicon chip. This elegant and scalable system has unique potential to transform the speed and cost of DNA sequencing. Oxford Nanopore also has collaborative projects in the development of solid state nanopores for further improvements in speed and cost. For further information please visit www.nanoporetech.com.
Contacts and sources: