Wenonah Vercoutere

Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel

RNA and DNA strands produce ionic current signatures when driven through an α-hemolysin channel by an applied voltage. Here we combine this nanopore detector with a support vector machine (SVM) to analyze DNA hairpin molecules on the millisecond time scale. Measurable properties include duplex stem length, base pair mismatches, and loop length. This nanopore instrument can discriminate between individual DNA hairpins that differ by one base pair or by one nucleotide.

Biosensors for DNA sequence detection.

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

Highly Accurate Classification of Watson-Crick Basepairs on Termini of Single DNA Molecules

We introduce a computational method for classification of individual DNA molecules measured by an alpha-hemolysin channel detector. We show classification with better than 99% accuracy for DNA hairpin molecules that differ only in their terminal Watson-Crick basepairs. Signal classification was done in silico to establish performance metrics (i.e., where train and test data were of known type, via single-species data files). It was then performed in solution to assay real mixtures of DNA hairpins. Hidden Markov Models (HMMs) were used with Expectation/Maximization for denoising and for associating a feature vector with the ionic current blockade of the DNA molecule. Support Vector Machines (SVMs) were used as discriminators, and were the focus of off-line training. A multiclass SVM architecture was designed to place less discriminatory load on weaker discriminators, and novel SVM kernels were used to boost discrimination strength. The tuning on HMMs and SVMs enabled biophysical analysis of the captured molecule states and state transitions; structure revealed in the biophysical analysis was used for better feature selection.

Sequence-dependent gating of an ion channel by DNA hairpin molecules.

DNA hairpins produce ionic current signatures when captured by the alpha-hemolysin nano-scale pore under conditions of single molecule electrophoresis. Gating patterns produced by individual DNA hairpins when captured can be used to distinguish differences of a single base pair or even a single nucleotide [Vercoutere,W.A. et al. (2003) Nucleic Acids Res., 31, 1311–1318]. Here we investigate the mechanism(s) that may account for the ionic current gating signatures. The ionic current resistance profile of conductance states produced by DNA hairpin molecules with 3–12 bp stems showed a plateau in resistance between 10 and 12 bp, suggesting that hairpins with 10–12 bp stems span the pore vestibule. DNA hairpins with 9–12 bp stems produced gating signatures with the same relative conductance states. Systematic comparison of the conductance state dwell times and apparent activation energies for a series of 9–10 bp DNA hairpins suggest that the 3′ and 5′ ends interact at or near the limiting aperture within the vestibule of the alpha-hemolysin pore. The model presented may be useful in predicting and interpreting DNA detection using nanopore detectors. In addition, this well-defined molecular system may prove useful for investigating models of ligand-gated channels in biological membranes.

Use of a Nanoscale Pore to Read Short Segments within Single Polynucleotide Molecules

Single-stranded polynucleotide molecules impede ionic current when they are driven through a nanoscale pore formed by the α-hemolysin heptameric channel (see J. Kasianowicz, D. Deamer, D. Branton, this volume). The duration and frequency of these blockades correlate with the length and concentration of the polymer examined. This suggests that translocation of RNA and DNA strands through the nanopore might also be used to derive a direct, high-speed readout of each molecule’s linear composition. We have recently shown that this is possible. Homopolymers of polycytidylic acid (poly C), polyadenylic acid (poly A), and polyuridylic acid (poly U) cause blockades of current through the α-hemolysin pore that are distinguishable from one-another based on amplitude and duration. These differences are due to the predominant secondary structure adopted by each homopolymer at room temperature in neutral buffer. We have also demonstrated that the nanopore instrument has sufficient sensitivity and resolution to detect short, discrete blocks within single polynucleotide molecules during translocation. For example, within an individual RNA strand, the transition from a 30-nucleotide poly A segment to a 70-nucleotide poly C segment can be read as an abrupt, 10 pA current change. A larger current change can also be observed at the transition between nucleotides and an abasic deoxyribose-phosphate segment inserted into a synthetic DNA strand. These polymers may be used to encode targeting molecules such as antibodies, gene specific oligonucleotides, and peptide agonists.

Fifteen Years of NASA Student Space Settlement Design Contests: Some Lessons

Since 1994, the NASA Ames Research Center has hosted an annual space settlement design contest for 6-12th grade students. Thousands of students and hundreds of teachers from around the world have involved themselves in space settlement, including environmental and life support systems, some devoting months of intense effort. Prize winners now find themselves at Harvard, Stanford, MIT and other top universities, and at least one flew a zero-gravity experiment for the European Space Agency (ESA). Contestants work at home and send their entries to NASA Ames each March. Extensive reference materials are supplied on the web. All entries are judged on a single day by a panel of NASA and contractor scientists and engineers. In 2007, the Ames center director, Pete Worden, was a judge. Many categories are created to generate a large number of winners and every attempt is made to reward entries that show serious effort with some sort of prize. All winners are invited to visit NASA Ames in June. This allows us to meet many of the best contestants and has led to a number of collaborations resulting in published technical papers. The contest is administered by a single, very part time individual, a public school teacher, at a total cost of

Discrimination among individual Watson–Crick base pairs at the termini of single DNA hairpin molecules

3-6 per contestant. Key lessons-learned include: