Nanopore sequencing is a technique in which DNA is driven electrophoretically through an orifice so small that each base must pass through one at a time. Translocation of thousands of bases of single stranded DNA has been demonstrated. If such long sequence runs could be read rapidly and accurately with no need for chemical reagents or the preparation of elaborate libraries, costs might be reduced to the point where personal genomes would become available for clinical use. Readouts based on the blockading of ion current have been able to resolve individual nucleotides and a single base trapped at a double-single strand junction in a hairpin but have not been able to read along a DNA molecule continuously. Very recently, we have shown that it is possible to identify individual bases and read along a DNA molecule using a technique we call Recognition Tunneling. Recognition molecules, covalently bound to electrodes, are used to transiently trap each base in turn through noncovalent bonds, giving distinct electronic signatures of all four bases and 5-methyl C. The trapping time with no external force applied to the DNA is long (seconds). However, unbinding is readily accelerated to very short times by the application of small forces, so Recognition Tunneling also provides a straightforward approach to translocation control. Here, we propose to combine Recognition Tunneling with nanopore translocation using metal or graphene nanopores, and metal or carbon nanotube reading electrodes, the probes and pores both being functionalized with recognition molecules. We will study translocation-control in functionalized, conducting nanopores, using both the bias across the pore and the surface potential of the conducting pore as control signals. Using a scanning-tunneling microscope (STM) platform, we will make measurements of Recognition Tunneling signals as DNA emerges from the nanopore. Multiscale (quantum to fluid-mechanical) simulations at Oak Ridge National Laboratory will help us to understand and optimize the translocation and readout processes. This understanding will be shared with collaborators who are developing nanopores with fixed (as opposed to STM) reading schemes with the ultimate goal of producing sequencing chips that are cheap and contain many thousands of devices.

Public Health Relevance

Recognition Tunneling is a new analytical tool that generates a distinct electronic signal for each of the four bases in DNA, as well as identifying a modification that underlies the epigenetic code. Here, we propose to use Recognition Tunneling to develop an instrument to read the sequence of DNA as it emerges from a nanopore. If successful, this instrument could reduce the costs of personal genomes and even enable epigenetic mapping of different tissues from the same individual.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
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Special Emphasis Panel (ZHG1-HGR-N (M2))
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Schloss, Jeffery
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Arizona State University-Tempe Campus
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Zhang, Bintian; Song, Weisi; Pang, Pei et al. (2017) Observation of Giant Conductance Fluctuations in a Protein. Nano Futures 1:
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