Session H7: Nanopore Biophysics

Interaction of DNA and Proteins with Single Nanopores

The bacterial toxins \textit{Staphylococcus aureus} alpha-hemolysin and \textit{Bacillus anthracis} protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. \newline \newline In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.   

Force measurements on a DNA molecule inserted into a solid-state nanopore

Single nanometer-sized pores (nanopores) in an insulating membrane are an exciting new class of nanosensors for rapid electrical detection of and actuation on single biomolecules. I will report (i) our fabrication of solid-state nanopores and translocation measurements of single dsDNA molecules through these pores, and (ii) our recent demonstration of measurements of the force acting on a single DNA molecule that is inserted in the nanopore. Ad (i): Siliconoxide nanopores are fabricated with single nanometer precision and direct visual TEM feedback. Translocation of double-strand DNA is monitored in the conductance of a voltage-biased pore. We find that DNA molecules can pass the pore both in a straight linear fashion and in a folded state. On molecules with a length from 3,000-100,000 base pairs, we observe a power-law scaling of the translocation time versus length, which we attribute to an effect of the hydrodynamic drag on the section of the polymer outside the pore. Measurements of DNA translocation at various salt concentrations reveal a crossover from a high-salt regime where current dips are seen, to a low-salt regime where current enhancements are observed. Ad (ii) For force measurements during the voltage-driven translocation of DNA and RNA, we have added an optical tweezer to our setup. With the tweezer, we hold a bead with a DNA molecule attached. Upon insertion of the DNA into the nanopore, the induced bead deflection yields a measure of the local force that acts on the DNA in the pore. The magnitude of the force involved is of fundamental importance in understanding and exploiting the translocation mechanism, yet so far has remained unknown. We obtain a value of 0.24 +/- 0.02 pN/mV for the force on a single DNA molecule, independent of salt concentration. Our data allow the first direct quantitative determination of the effective DNA charge of 0.53 +/- 0.05 electrons per base pair, corresponding to a 73{\%} reduction of the bare DNA charge. Our novel single-molecule technique for local force sensing and actuation bears great promise for biophysical studies, e.g. for the study of DNA-protein binding or unfolding of RNA.   

Protein unraveling through a single protein nanopore

The ability to respond to an external stimulus is a fundamental process in living systems. Based on this principle, we were able to design an unusual temperature-responsive pore-based nanostructure with a single movable elastin-like-peptide (ELP). The peptide is placed within the cavity of the alpha-Hemolysin protein pore. The temperature-dependent properties of single engineered pores were monitored by single-channel current recording in planar lipid bilayers. If a voltage bias was applied, the engineered pores exhibited transient current blockades, the nature of which depended on the length and sequence of the inserted ELP. These blockades are associated with the peptide excursions into the narrowest region of the pore. At low temperatures, the ELP is fully expanded and blocks reversibly and completely the pore. At high temperatures, the ELP is dehydrated and structurally collapsed, thus enabling a substantial ionic flow. Potential applications of this nanostructure in several arenas will be discussed.   

Detecting Single DNA and Proteins Using a Solid-state Nanopore Device

Charged single proteins and DNA molecules can be detected as they are driven through a solid-state nanopore by an applied electric field. The solid-state nanopores are fabricated in a free standing silicon nitride membrane using low energy noble gas ion beams. We demonstrate the silicon nitride nanopore based sensor can measure the properties of single molecules of proteins, double stranded DNA, and single stranded DNA molecules at different temperature, pH, and ionic strength. This technique allows us to discriminate between different types of molecules, different conformations of the same molecules, and also determine the configuration of individual molecules as well as their configuration distribution. We demonstrate the silicon nitride nanopore sensing system is robust and capable of detecting structural information of protein and DNA at extreme conditions.   

Microscopic Kinetics of DNA Translocation through Synthetic and Biological Nanopores

Using highly focused electron beams, artificial pores of nanometer diameters can be manufactured in ultra-thin silicon membranes with a sub-nanometer precision. A trans-membrane voltage bias can drive DNA strands through such pores; the resulting electrical signals can be recorded. As the diameter of the pore as well as the thickness of the silicon membrane can be made to match precisely the dimensions of a DNA nucleotide, the electrical signals produced by the interaction of DNA with the pore were proposed to contain information about the DNA sequence. In order to relate the DNA sequence to the measured electrical signals we characterized DNA conformations inside the pore through molecular dynamics simulations. A typical simulated system included a patch of a silicon membrane dividing electrolyte solution into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. To calibrate our methodology, we carried out MD simulations of DNA translocation through an $\alpha $-hemolysin channel suspended in a lipid bilayer. Our results suggest that the rate-limiting step for DNA translocation through narrow synthetic pores is not the actual transit of DNA, but rather the search for such initial conformation that facilitates subsequent translocation. At the same time, hydrophobic adhesion of DNA bases to the pore walls may considerably slow down or halt DNA translocation. We observed a threshold electric field for translocation of double stranded DNA through pores smaller in diameter than a DNA double helix occurring due to the overstretching transition at load forces of $\sim $60 pN. In narrow pores, DNA bases were observed to tilt collectively towards the 5'-end of the strand, which explains experimentally observed directionality of single stranded DNA in the transmembrane pore of $\alpha $-hemolysin.