Feedback control of a DNA molecule tethered in a nanopore to repeatedly probe DNA-binding enzymes.
This paper demonstrates feedback voltage control of a single DNA molecule tethered in a biological nanopore. The nanopore device monitors ionic current through a single protein pore inserted in a lipid bilayer. The limiting aperture of the pore is just sufficient (1.5 nm diameter) to accommodate single-stranded DNA. The tethered DNA is double stranded on each end, with a single stranded segment that traverses the pore. Voltage control is used to regulate the motion of the tethered DNA, for repeated capture and subsequent voltage-promoted dissociation of DNA-binding enzymes above the nanopore. In initial experiments using the Klenow fragment of Escherichia coli DNA polymerase I, control of 8 independent tethered DNA molecules yielded 337 dissociation events in a period of 380 seconds. The resulting distribution of DNA-protein dissociation times can be used to model the free energy profile of dissociation. Moreover, the approach is applicable to numerous enzymes that bind or modify DNA or RNA including exonucleases, kinases, and other polymerases.
Wilson
Noah A
NA
Baskin School of Engineering, University of California, Santa Cruz, 1156 High Street, CA, USA. noahness@soe.ucsc.edu
U-Shumays
Robin Ab
RA
Lieberman
Kate
K
Akeson
Mark
M
Dunbar
William B
WB
D004247:DNA / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005246:Feedback; D008051:Lipid Bilayers / Q000737:chemistry; D008846:Micromanipulation / Q000295:instrumentation* / Q000379:methods; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D036103:Nanotechnology / Q000295:instrumentation* / Q000379:methods; D016062:Porosity; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D008051:Lipid Bilayers; D004247:DNA; D004259:DNA-Directed DNA Polymerase