The proposed research program is an integrated collaborative effort between 2 laboratories experienced in nanopore research, protein engineering, and molecular recognition. The proposal addresses experimentally the most fundamental and critical issues in the field of single-molecule DNA sequencing by the protein nanopore approach, namely the nanopore itself, nucleobase recognition, and the moderation of ss-DNA transit times through the nanopore. The proposed work will establish protein nanopore technology for short (<1000 base) reads at a considerable price reduction. It is an important step on the path to accurate high-speed genome sequencing at greatly reduced cost.
The specific aims of the proposed research program are: (1) Genetically engineered a-hemolysin pores for base recognition. A constriction will be formed within the pore at which a single base confronts a ring of amino acid side chains generated by mutagenesis. The interaction restricts the current flow through the pore and the residual current differs for each base. (2) Chemically modified pores for base recognition. Natural nucleobases and unnatural analogues will be attached at specific sites within the pore. The modifications will provide base recognition and act as molecular brakes to slow the DNA transit time. (3) Attached enzymes to control translocation. Unidirectional DNA transit through the pore will be controlled by DNA polymerases so that sequence determination by amplitude-based recognition can be optimized. (4) Additional improvements to the nanopore through protein engineering. We will examine: (i) Control of the orientation of DNA within the pore with a molecular slide, (ii) Polymer-filled pores to slow DNA transit;(iii) Engineered pores other than a-hemolysin;(iv) Molecular adapters for base recognition. (5) Multipass reading with rotaxanes. DNA trapped as a supramolecular rotaxane can be moved back and forth in the pore by switching the applied potential, allowing multipass sequencing of DNA strands with reduced error rates. (6) Manipulation of the physical conditions. Nucleic acids contain secondary structure. The threading of ss-DNA at high temperatures or from denaturants will improve reads and prevent permanent blockades of the pore.
| Ayub, Mariam; Bayley, Hagan (2016) Engineered transmembrane pores. Curr Opin Chem Biol 34:117-126 |
| Stoddart, David; Franceschini, Lorenzo; Heron, Andrew et al. (2015) DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing. Nanotechnology 26:084002 |
| Bayley, Hagan (2015) Nanopore sequencing: from imagination to reality. Clin Chem 61:25-31 |
| Lee, Joongoo; Bayley, Hagan (2015) Semisynthetic protein nanoreactor for single-molecule chemistry. Proc Natl Acad Sci U S A 112:13768-73 |
| Huang, Shuo; Romero-Ruiz, Mercedes; Castell, Oliver K et al. (2015) High-throughput optical sensing of nucleic acids in a nanopore array. Nat Nanotechnol 10:986-91 |
| Ayub, Mariam; Stoddart, David; Bayley, Hagan (2015) Nucleobase Recognition by Truncated ?-Hemolysin Pores. ACS Nano 9:7895-903 |
| Stoddart, David; Ayub, Mariam; Höfler, Lajos et al. (2014) Functional truncated membrane pores. Proc Natl Acad Sci U S A 111:2425-30 |
| Rosen, Christian B; Rodriguez-Larrea, David; Bayley, Hagan (2014) Single-molecule site-specific detection of protein phosphorylation with a nanopore. Nat Biotechnol 32:179-81 |
| Rodriguez-Larrea, David; Bayley, Hagan (2014) Protein co-translocational unfolding depends on the direction of pulling. Nat Commun 5:4841 |
| Clamer, Massimiliano; Höfler, Lajos; Mikhailova, Ellina et al. (2014) Detection of 3'-end RNA uridylation with a protein nanopore. ACS Nano 8:1364-74 |
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