My current research in collaboration with David Deamer has two foci:
- The use of nanopore detectors to understand the dynamics and structure of DNA duplex ends, including those of retrotransposons and HIV
- The coupling of processive DNA-modifying enzymes to nanopores, both protein and solid-state
Nanopore detectors are instruments built around a tiny pore in a membrane or thin, solid-state wafer. The pore is just big enough to allow a single strand of DNA to pass through. A voltage applied across the membrane generates an ionic current capable of pulling DNA molecules (which are negatively charged) through the pore. When the DNA molecule blocks the opening of the nanopore, it causes a characteristic decrease in the current, allowing discrimination between individual DNA molecules. A computer trained by machine-learning techniques recognizes the signals generated by different DNA molecules.
For example, our most advanced instrument is based on a single nanoscale pore formed by the bacterial toxin, alpha-hemolysin. When individual DNA hairpin molecules are captured by an applied electric field, the duplex stem is suspended in the pore vestibule (Nature Biotechnol 2001 Mar; 19(3):248-52). We have shown that the duplex stems impede the monovalent ionic current through the pore in a sequence-dependent manner (Nucleic Acids Res 2003 Feb 15; 31(4):1311-8 and Biophys J 2003 Feb; 84(2 pt 1):967-76). Early results suggest that low-frequency noise at one of these current levels reports on structural changes of the helix terminus of one duplex molecule. Using machine-learning tools, these differences can be identified in real time and in less than 50 milliseconds. One of the great strengths of this approach is that a comprehensive set of dynamic and structural data for all permutations of 6-base-pair DNA termini (i.e., 46 = 4096 members) could be examined in a few months. This would be entirely unfeasible using X-ray crystallography or NMR spectroscopy alone.
- MORE...nanopore analysis of DNA molecules
- MORE...nanopore project at UCSC
- MORE...nanopore project members and collaborators
Nanopore Detectors with Processive DNA-Modifying Enzymes
The second new focus concerns coupling of processive DNA-modifying enzymes to the alpha-hemolysin pore. Recent experiments I undertook with Andrea Solbrig, Veronica DeGuzman, and Amy Coombs have proven that we can couple nanopore detection of single DNA or RNA molecules with lambda exonuclease, exonuclease I from E. coli, and polynucleotide phosphorylase (PNP). This is a first, and it opens entirely new avenues of inquiry concerning single enzyme function and its application in DNA technology.