Fig 1.
Single-molecule sensing with a nanopore device.
(a) Schematic diagram of the setup with voltage V applied across a single nanopore fabricated in a solid-state substrate, while measuring the current through the pore. (b) A representative event caused by a 3.2 kb dsDNA passing through an 27 nm diameter nanopore at V = 100 mV (1M LiCl). Events are quantitated by shift conductance (δG = δI/V) and duration. (c) Scatter plot of δG versus duration for 713 events recorded over 10 minutes.
Fig 2.
The bisPNA probe binds to dsDNA in conditions compatible with nanopore experiments, with and without a PEG payload.
(a) The cysteine substituted bisPNA (black, U-shape) is bound to 324 bp scaffold dsDNA (blue) making a triplex helix. A modified cytosine is used that has less pH dependence when making Hoogsteen contacts (J). The two halves of the PNA are separated by a flexible PEG linker (O) that has a cysteine (C) amino acid in the middle. Lysines (K) are added at each end to increase the stability. PEGs containing maleimides react with the cysteine residues (C) in the PNA creating the DNA/bisPNA-PEG complex. (b) The DNA/bisPNA complex was found to be stable in up to 1M LiCl over a half-hour incubation at room temperature, indicating that the majority of the complex will be intact throughout the duration of the nanopore assay. Image shows 10% PAGE EMSA with lanes: 1) high molecular weight ladder, 2) DNA (324 bp), 3) DNA (negative control with scrambled 7 bp target sequence) with PNA, 4) DNA (negative control) with PNA-PEG (5 kDa), 5) DNA/PNA-PEG (5 kDa) and 10) DNA/PNA. Lanes (6-9) are DNA/PNA-PEG (5 kDa) after 30 min incubation in increasing LiCl concentrations (0.25, 0.5, 0.75, 1M).
Fig 3.
Short DNA and DNA/bisPNA events were indistinguishable in a ∼7 nm pore.
(a) Population of δG vs. duration for all events in experiments with DNA alone (10 nM, 100 and 200 mV) and DNA/bisPNA (10 nM, 100 mV) show that most events are too fast (left of the 24 μs resolution, green dashed line) to resolve full amplitude depth. (b) Event δG histogram for all events at least 24 μs in duration shows that DNA and DNA/bisPNA are indistinguishable at 100 mV. (c) Duration histogram and representative events for DNA and DNA/bisPNA at 100 mV. (d) On the natural-log scale, the fraction of open channel times faster than time T for the three data sets are shown, with the data fit by a straight line (single exponential probability distribution, Methods).
Fig 4.
A single bisPNA on DNA is resolvable with a ∼6 nm pore.
(a) Population of δG vs. duration for all events in the experiment with DNA/bisPNA (22 nM, 100 mV) with the ∼6 nm pore, overlaid with the DNA/bisPNA data from Fig 3. The event δG histogram (b) and duration histogram (c) shift significantly by using the smaller pore. (d) Schematics of the nanopores and the complex, all sized using a common scale for visual comparison, and a representative event from each experiment (reporting δG, duration). The bisPNA creates a bulge on DNA approximately ∼4 nm in width and length.
Fig 5.
A single bisPNA on DNA is resolvable with a 17-21 nm pore by adding bulk through bisPEG-PNA linking.
(a) Representative events: DNA/bisPNA (left), and DNA/bisPNA-PEG with up to 3 PEGs on each PNA, and PEG sized 5 kDa (middle) and 10 kDa (right). Molecule depictions show linear PEG and DNA sized to scale for visual comparison. (b) Population of δG vs. duration for all events in each data set, each at 2 nM and 100 mV in 1 M LiCl, with δG histogram (c) and duration histogram (d) shifted by adding PEGs of increasing size. (e) Evolution of the modeled nanopore diameter, using the time history of the open channel conductance (S2 Section), spanning 30 min (DNA/bisPNA), 58 minutes (DNA/bisPNA-PEG 5 kDa) and 40 minutes (DNA/bisPNA-PEG 10 kDa).
Fig 6.
The 300 bp DNA/γPNA-PEG 5 kDa complex is resolvable with a 26-36 nm diameter pore in 100 mM LiCl, providing positive detection of the CFTRΔF508 gene mutation.
(a) Representative events with the pore initially at 26 nm in diameter, reporting δG and duration values. (b) Population of δG vs. duration for all 221 events over 52 minutes at 1 nM complex and 200 mV. Events span three pore sizes (26 nm, 32nm, 36 nm) that were enlarged by dielectric breakdown. The green line (24 μsec) is the minimum duration for resolving δG. (c) δG histogram and (d) duration histogram of all events. (e) 5% PAGE EMSA shows the 22 bp γPNA-PEG (5k) bound to the 300 bp DNA at the 22 bp target sequence that encompasses the CFTRΔF508 mutation (right lane). The γPNA-PEG (5k) does not bind to the 300 bp DNA that has the wild-type (i.e., non-mutant) sequence (middle lane), showing target specificity. The sizing ladder is low molecular weight (left lane).