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.

Figure 4. Comparison of single-mismatch detection with gold-quenched beacons versus DABCYL-quenched beacons. Titration of 5 µM of random target mixed with 4.2 nM of gold-DNA-rhodamine 6G conjugate and 0.6 µM of gold (A), and 5 µM of random target mixed with 10 nM of molecular beacon (B), with the perfect target (target 2) and the mismatch one (target 3). Target concentrations vary from 67 pM to 13 µM. For both probes, the perfect target (solid line) produces a faster and sharper increase of fluorescence than the target containing the mismatch (dashed line). Fluorescence intensities due to the buffer and the gold have been subtracted. The inset graphs in (A) and (B) show the evolution of the fluorescence as a function of time when the probe is mixed with 5 µM of random targets. In both cases, the random targets do not induce any change of fluorescence of the probe during the time of the titration. The hybridization is thus very specific to the matched or the mismatched targets. (C) Ratio between the titration curve with the perfect target (target 2) and the titration curve with the mismatched one (target 3). (D) Resolution of a matched and a mismatched target, competing for hybridization. Molecular beacon (dashed line), gold-DNA-dye conjugate (solid line). α is the population ratio of match to mismatch targets. The concentration of perfect target is fixed at 0.2 µM. D

We previously reported that 5'-mononucleotides organized within a multilamellar lipid matrix can produce oligomers in the anhydrous phase of hydration-dehydration (HD) cycles. However, hydrolysis of oligomers can occur during hydration, and it is important to better understand the steady state in which ester bond synthesis is balanced by hydrolysis. In order to study condensation products of mononucleotides and hydrolysis of their polymers, we established a simulation of HD cycles that would occur on the early Earth when volcanic land masses emerged from the ocean over 4 billion years ago. At this stage on early Earth, precipitation produced hydrothermal fields characterized by small aqueous pools undergoing evaporation and refilling at elevated temperatures. Here, we confirm that under these conditions, the chemical potential made available by cycles of hydration and dehydration is sufficient to drive synthesis of ester bonds. If 5'-mononucleotides are in solution at millimolar concentrations, then oligomers resembling RNA are synthesized and exist in a steady state with their monomers. Furthermore, if the mononucleotides can form complementary base pairs, then some of the products have properties suggesting that secondary structures are present, including duplex species stabilized by hydrogen bonds.

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.