Research published this week in JACS shows continuous and controlled translocation of a single stranded DNA (ssDNA) polymer through a protein nanopore by a DNA polymerase enzyme.
The paper by researchers at the University of California Santa Cruz (UCSC) provides the foundation for a molecular motor, an essential component of Strand Sequencing using nanopores. Researchers at UCSC are collaborating with the UK-based company Oxford Nanopore Technologies, developers of a nanopore DNA sequencing technology.
The new research advances previous work showing that DNA could be moved through a nanopore using a polymerase. DNA movement in the previous study was performed by a series of polymerases and required complex electronics for control. Improvements noted in the JACS paper include techniques to allow continuous ssDNA movement, giving an uninterrupted signal as the strand was moved through the nanopore in real time. The enzyme-nanopore construct was active and measurable in a constant electronic field without complex electronics.
Controlled initiation of the polymerase processing at the site of the nanopore-enzyme complex allowed sequential measurement of multiple ssDNA molecules using a single experimental setup . Furthermore the polymerase exhibited tenacious binding with the DNA polymer, unlike previous enzymes researched in similar conditions. These results demonstrate that qualities of the phi29 DNA polymerase are commensurate with a strand sequencing technology.
In the 'strand sequencing' method of nanopore DNA sequencing, ionic current through a protein nanopore is measured and current disruptions used to identify bases on a ssDNA polymer in sequence, as it translocates the pore. Two key challenges for this method are: engineering a nanopore to enable identification of individual bases when a ssDNA polymer spans the pore and a mechanism for controlling translocation of ssDNA at a consistent and appropriate speed to enable base identification through electronic measurements. Translocation techniques described in this paper are compatible with base identification technology being performed in the laboratories of Oxford Nanopore Technologies and its collaborators.
"This work with the phi29 polymerase has allowed us to make important progress on a key element of DNA strand sequencing," said investigator Professor Mark Akeson of the University of California, Santa Cruz. "While previous work showed that translocation control was possible in theory, this work shows that DNA translocation control is achievable in conditions that are compatible with an electronic sequencing technology. We look forward to further collaboration with Oxford Nanopore to realise this research."
"The 'strand sequencing' method of DNA sequencing using a nanopore has been studied for many years, but this paper shows for the first time that DNA can be translocated by an enzyme using methods that are consistent with a high throughput electronic technology," said Dr Gordon Sanghera, CEO of Oxford Nanopore. "We are excited by this work and its potential when coupled with additional recent developments in DNA base identification on DNA strands, the other critical element for strand sequencing."
Notes to Editors
Reference: Processive Replication of Single DNA Molecules in a Nanopore Catalyzed by phi29 DNA Polymerase (subscription needed). Available online at http://pubs.acs.org/doi/abs/10.1021/ja1087612 (subscription needed for full article)
Work conducted in this paper
In this JACS paper, single stranded DNA (ssDNA) was translocated through an alpha hemolysin nanopore using the enzyme bacteriophage phi29 DNA polymerase (phi29DNAP). The enzyme had been chosen due its favourable processivity and high binding strength with DNA substrates. The nanopore-enzyme construct was shown to be stable in an electric field under a 180 mV applied potential, a level that is compatible with simultaneous identification of DNA bases using a nanopore. In the presence of deoxynucleoside triphosphates, processing of the ssDNA could be initiated specifically at the nanopore, with real time addition of nucleotides resulting in ssDNA translocation through the pore. The nanopore used in this research was not engineered for nucleotide discrimination and therefore an abasic area within the ssDNA was introduced to allow observation of its passage through the pore.
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 methods (1, 2) to correctly identify individual nucleotides on ssDNA immobilised within an AHL nanopore and to identify modified bases on a DNA strand. Further work continues at Oxford Nanopore and in the laboratories of the Company's collaborators and this work is compatible with the methods described in the JACS paper.
Oxford Nanopore Technologies Ltd
Oxford Nanopore Technologies Ltd is developing a revolutionary technology for direct, electronic 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. In exonuclease sequencing, individual bases are cleaved from a strand of DNA and identified as they pass through a protein nanopore. In strand sequencing a DNA polymer is analysed as it translocates the nanopore. 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.
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