To identify and discriminate alleles, Affymetrix currently uses a hybridization technique in conjunction with microarrays on high-throughput platforms to measure the difference in thermal stability between matched and mismatched target-probe pairs of dsDNA. We plan to use a related strategy for detecting SNPs targets that leverages the stretching transition. Our strategy employs short (10-12 nucleotide long) probes hybridized to the target cognate sites on ssDNA and uses high-speed SMFS to measure the threshold voltage associated with stretching or unzipping the templates in a pore <2.5nm in diameter. The transition from dsDNA to ssDNA, called the helix-coil transition, is a key element of this strategy. SMFS has been used to analyze the helix-coil transition, revealing a dichotomy in the force required to dissociate base pairs: the force is different depending on whether the DNA is unzipped by pulling parallel to the bases, or stretched by pulling transverse to the base-pairs. About F_unzip = 10 - 30 pN of force is required to unzip dsDNA. On the other hand, when dsDNA is stretched beyond its contour length, it undergoes a cooperative stretching transition near F_str = 60 - 70 pN. This overstretching transition has been interpreted as force-induced melting in which the two DNA strands break apart and unwind. Synthetic nanopores can be used to accomplish both measurements because large forces can be applied using the field in the pore, and because the pore diameter can be controlled with sub-nanometer precision.

We studied the helix-coil transition by measuring the permeability of hairpin DNA (hpDNA) through a nanopore in a silicon nitride membrane. Nucleic acid hairpins or stem-loops, formed from self-complementary sequences, are found regularly in DNA and RNA secondary structure. The dynamics of hairpin structure has already been carefully scrutinized with SMFS accomplished using α-HL. The structure is not static; there is a folded (closed) conformation consisting of a single-stranded coil, a double-stranded stem and a coil, and an unfolded (open or melted) state. The folded (closed) conformation is characterized by a low enthalpy due to base pairing in the stem, while the open state has high entropy due to the large number of configurations.available to ssDNA. Nanopore SMFS is superior to other methods because it can done without a molecular tether or anchor, and the loading rate or the force can be held constant and range up to 100Vnsec, while maintaining high throughput (~1000 molecules/second). But unlike α-HL, a synthetic nanopore affords us the opportunity to constrain the transverse motion of the molecule through control of the diameter.

We systematically investigated hpDNA permeability through a membrane as a function of the applied voltage and pore diameter. To determine if hpDNA permeates the membrane through the pore, we analyzed the extract taken from the anode using qPCR. We observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter. We assume that the threshold voltage corresponds to the minimum force required to impel a hairpin through the pore. Assuming that the stem frustrates the permeation of hpDNA, a large force corresponding to the change in free energy, ΔG, of the helix-coil transition will be required to dissociate the bases and induce the translocation of DNA.

We have found a dependence of the threshold voltage on the pore diameter; the threshold voltage collapses from about ~2V to ~0.5V as the pore size decreases from 1.5nm to 1nm, indicative of a dramatic change (x4) in the force required to induce the transition. Assuming that a pore with d<1.5nm excludes dsDNA and precludes stretching, we attribute this change to the difference between stretching and unzipping DNA. Ostensibly, the force required to dissociate base-pairs is different depending on whether the DNA is unzipped by pulling parallel to the bases or stretched by pulling transverse to the base-pairs. It takes less force to unzip DNA than to stretch the backbone. With the hpDNA's conformation unconstrained by the pore walls, the bases can rotate so that much of the force is applied along the axis of the hydrogen bonds connecting the bases. And so the helix-coil transition occurs by unzipping. However, if the hairpin penetrates deep within the pore, only a small component of the force is directed along the axis of the hydrogen bonds.

In summary, we observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the DNA. For a diameter 1.5<d<2.3nm the threshold corresponds to the force required to stretch the stem of the hairpin, while for 1.0<d<1.5 nm, the threshold collapses because the stem unzips with a lower force than required for stretching. In related work, we have observed a threshold voltage for the rupture of the bond between a restriction enzyme and DNA that can be used to discriminate single nucleotide polymorphisms. Although speculative, it seems likely that the threshold for a hairpin to permeate the pore is related to the free energy and molecular stability, but an unambiguous interpretation requires knowledge of the molecular configuration in the pore: i.e. whether the molecule enters oriented coil- or loop-first. This information could be recovered through force spectroscopy studies of the translocation of hairpins one at a time through the pore, but first we have to sort out the relationship between the current transients and the configuration of the molecule in the pore.