Dynamics of DNA Molecules in a Membrane Channel Probed by Active Control Techniques

Abstract

The dynamics of single-stranded DNA in an alpha-Hemolysin protein pore was studied at the single-molecule level. The escape time for DNA molecules initially drawn into the pore was measured in the absence of an externally applied electric field. These measurements revealed two well-separated timescales, one of which is surprisingly long (on the order of milliseconds). We characterized the long timescale as being associated with the binding and unbinding of DNA from the pore. We have also found that a transmembrane potential as small as 20 mV strongly biased the escape of DNA from the pore. These experiments have been made possible due to the development of a feedback control system, allowing the rapid modulation of the applied force on individual DNA molecules while inside the pore.

Nucleic acid transport through carbon nanotube membranes

“…With some uncertainty, caused by the small number of events at low fields, we find that the characteristic time of the slow decay in the exit-time distributions seems to decrease exponentially with the applied electric field. This result is consistent with the experimental observations of Bates et al (5). Based on Eq.…”

“…Exit-time distributions similar to those obtained here in the simulations and predicted by the trap-diffusion model of Eqs. 1 and 2 were observed in recent single-molecule measurements of single-stranded DNA exiting from membraneinserted ␣-hemolysin channels (5). The approximately exponential tail after a rapid initial burst in the measured exit times was attributed to pore-binding DNA populations, consistent with our observations and model.…”

“…The electric field in typical nucleic acid translocation experiments with membrane-inserted ␣-hemolysin channels is in the range of 0.02-0.04 V͞nm (120 mV across 3-to 5-nm membrane thickness) (2,5), which is Ϸ20 times smaller than the smallest estimated electric field in our nanotube membrane simulations. Use of relatively high electric fields was necessary to induce at least nanometer-scale motion on the nanosecond simulation time scales.…”

“…To analyze the resulting multiphasic kinetics, we use a trap-diffusion model that combines diffusive RNA motion along the pore with random trapping. We will show that the model motivated by our simulations can also account for detailed experimental measurements of DNA translocation through ␣-hemolysin pores (5). By varying the strength of the transmembrane potential, we will study translocation of RNA at different driving forces.…”

“…Electrostatic membrane potentials play a critical role in biopolymer transport, as demonstrated for the import of unfolded proteins into the mitochondrial matrix (1). Electrostatically driven membrane translocation is also increasingly used to measure the properties of single polymers (2)(3)(4)(5)(6). The blockage of ionic currents during electric-field-driven translocation of individual nucleic acid molecules through membrane-inserted ␣-hemolysin channels (7) was shown to depend on length, base composition, and sequence (2,3), suggesting possible applications in ultrafast and single-molecule sequencing of nucleic acids.…”