The structure and dynamics of DNA duplex ends can influence numerous enzyme-dependent processes such as transposition of phage Mu, and integration of HIV dsDNA copies into target chromosomal DNA. Despite their critical importance in biology, DNA duplex ends are significantly under-represented in NMR and crystal structure studies, and their dynamic properties have not been measured directly. The long-term goal of this project is to establish a comprehensive dynamic and structural map of DNA duplex ends. This map will be at near angstrom precision and will include all possible combinations of base pairs for the last six positions of the DNA helix (i.e. 46 = 4096 unique sequences). The approach we will use to generate the map couples NMR spectroscopy with an instrument developed in our laboratory for single molecule measurements. This instrument is based on a nanoscale pore formed by the bacterial toxin, ?-hemolysin. When individual DNA hairpins are captured in this pore by an applied electric field, the duplex stem is suspended in the pore vestibule. Preliminary results suggest that low frequency (kHz) current noise during DNA capture is caused by structural changes of the helix terminus.
The aim of this R-21 proposal is to determine if the nanopore detector gives unbiased reads of DNA duplex end structure and dynamics in the Hz to MHz range. Specific questions we will address include: 1) To what extent does the pore vestibule and the applied electric field influence DNA dynamics? 2) Are the duplex end kinetics independent of hairpin loop identity and duplex stem length? 3) There is broad consensus that some sequences are unusually rigid (e.g. A tracts) while others are highly flexible (e.g. TATA). Do these sequences cause nanopore current signatures that cluster together as predicted? 4) Can pattern recognition algorithms be used to automate data analysis? We consider this research to be suited for R-21 funding because there is substantial risk that the outcome will be negative, i.e. we may find that the observed current dynamics are only relevant to the limited case of DNA hairpins captured in a nanoscale protein cavity. However, if the research is successful, it will yield a new way to observe DNA dynamics that reports precise kinetic data, and that is amenable to high throughput experiments. These nanopore experiments will be used to direct subsequent NMR experiments and vice versa. ? ?
| Hurt, Nicholas; Wang, Hongyun; Akeson, Mark et al. (2009) Specific nucleotide binding and rebinding to individual DNA polymerase complexes captured on a nanopore. J Am Chem Soc 131:3772-8 |
| Churbanov, Alexander; Winters-Hilt, Stephen (2008) Clustering ionic flow blockade toggles with a mixture of HMMs. BMC Bioinformatics 9 Suppl 9:S13 |
| Benner, Seico; Chen, Roger J A; Wilson, Noah A et al. (2007) Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nat Nanotechnol 2:718-24 |
