Monitoring the escape of DNA from a nanopore using an alternating current signal.

We present the use of an alternating current (AC) signal as a means to monitor the conductance of an alpha-hemolysin (alphaHL) pore as a DNA hairpin with a polydeoxyadenosine tail is driven into and released from the pore. Specifically, a 12 base pair DNA hairpin attached to a 50-nucleotide poly-A tail (HP-A(50)) is threaded into an alphaHL channel using a DC driving voltage. Once the HP-A(50) molecule is trapped within the alphaHL channel, the DC driving voltage is turned off and the conductance of the channel is monitored using an AC voltage. The escape time, defined as the time it takes the HP-A(50) molecule to transport out of the alphaHL channel, is then measured. This escape time has been monitored as a function of AC amplitude (20 to 250 mV(ac)), AC frequency (60-200 kHz), DC drive voltage (0 to 100 mV(dc)), and temperature (-10 to 20 degrees C), in order to determine their effect on the predominantly diffusive motion of the DNA through the nanopore. The applied AC voltage used to monitor the conductance of the nanopore has been found to play a significant role in the DNA/nanopore interaction. The experimental results are described by a one-dimensional asymmetric periodic potential model that includes the influence of the AC voltage. An activation enthalpy barrier of 1.74 x 10(-19) J and a periodic potential asymmetry parameter of 0.575 are obtained for the diffusion at zero electrical bias of a single nucleotide through alphaHL.

Multimodal neuroelectric interface development.

We are developing electromyographic and electroencephalographic methods, which draw control signals for human-computer interfaces from the human nervous system. We have made progress in four areas: 1) real-time pattern recognition algorithms for decoding sequences of forearm muscle activity associated with control gestures; 2) signal-processing strategies for computer interfaces using electroencephalogram (EEG) signals; 3) a flexible computation framework for neuroelectric interface research; and d) noncontact sensors, which measure electromyogram or EEG signals without resistive contact to the body.

Creating a Single Sensing Zone within an Alpha-Hemolysin Pore Via Site Directed Mutagenesis.

Although significant progress has recently been made towards realizing the goal of direct nanopore based DNA sequencing [1], there are still numerous hurdles that need to be overcome. One such hurdle associated with the use of the biological nanopore α-hemolysin (αHL) is the fact that the wild type channel contains three very distinct recognition or sensing regions within the ß-barrel [2, 3], making identification of the bases residing within or moving through the pore very difficult. Through site directed mutagenesis, we have been able to selectively remove one of two sensing regions while simultaneously enhancing the third. Our approach has led to the creation of αHL pores containing single sensing zones and provides the basis for engineering αHL pores suitable for direct DNA sequencing.

The potential and challenges of nanopore sequencing.

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.