The objective of this project is to engineer a new protein pore, MspA, for nanopore DNA sequencing. MspA's short and narrow constriction, its extreme stability against denaturation and its tolerance to mutations make this protein an ideal, inexpensive and novel nanopore sequencing development platform. We have obtained exciting results that demonstrate the feasibility of our proposal. We designed and made MspA mutants that pass DNA. Importantly, mutated MspA can already nearly resolve single nucleotides using co-passing current alone. Molecular dynamics simulation of MspA agrees excellently with experiment. A prototype fast, low-noise current amplifier was built specifically for nanopore sequencing experiments.
Our specific aims are to (i) rationally design, produce and test MspA mutants to improve DNA base recognition and reduce translocation speed;(ii) use molecular dynamics simulation to understand how DNA interacts with MspA and to optimize MspA for nanopore sequencing;(iii) construct a single chain protein to further improve DNA base sensitivity and control of DNA motion in an asymmetric MspA pore;(iv) construct a highly sensitive electronic amplifier and a practical bilayer apparatus. We have formed a team of three outstanding labs with complementary expertise in protein science, protein simulation, single-channel experiments, molecular biology, and instrumentation to realize these aims. It is our goal to develop a system that can sequence a human genome for under $1000.

Public Health Relevance

This three university team is engineering a novel pore from mycobacteria, MspA, for nanopore DNA sequencing. MspA has an ideal shape for nanopore sequencing. The protein pore is remarkably tolerant of mutations so that it can be exactly tailored to be sensitive to individual nucleotides when DNA passes through it.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project (R01)
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Special Emphasis Panel (ZHG1-HGR-N (M1))
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Schloss, Jeffery
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University of Washington
Schools of Arts and Sciences
United States
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Pavlenok, Mikhail; Niederweis, Michael (2016) Hetero-oligomeric MspA pores in Mycobacterium smegmatis. FEMS Microbiol Lett 363:
Comer, Jeffrey; Aksimentiev, Aleksei (2016) DNA sequence-dependent ionic currents in ultra-small solid-state nanopores. Nanoscale 8:9600-13
Bhattacharya, Swati; Yoo, Jejoong; Aksimentiev, Aleksei (2016) Water Mediates Recognition of DNA Sequence via Ionic Current Blockade in a Biological Nanopore. ACS Nano 10:4644-51
Laszlo, Andrew H; Derrington, Ian M; Gundlach, Jens H (2016) MspA nanopore as a single-molecule tool: From sequencing to SPRNT. Methods 105:75-89
Craig, Jonathan M; Laszlo, Andrew H; Derrington, Ian M et al. (2015) Direct Detection of Unnatural DNA Nucleotides dNaM and d5SICS using the MspA Nanopore. PLoS One 10:e0143253
Derrington, Ian M; Craig, Jonathan M; Stava, Eric et al. (2015) Subangstrom single-molecule measurements of motor proteins using a nanopore. Nat Biotechnol 33:1073-5
Laszlo, Andrew H; Derrington, Ian M; Ross, Brian C et al. (2014) Decoding long nanopore sequencing reads of natural DNA. Nat Biotechnol 32:829-33
Chaudhry, Jehanzeb Hameed; Comer, Jeffrey; Aksimentiev, Aleksei et al. (2014) A Stabilized Finite Element Method for Modified Poisson-Nernst-Planck Equations to Determine Ion Flow Through a Nanopore. Commun Comput Phys 15:
Ross, Brian C (2014) Mutual information between discrete and continuous data sets. PLoS One 9:e87357
Maffeo, C; Yoo, J; Comer, J et al. (2014) Close encounters with DNA. J Phys Condens Matter 26:413101

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