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

Abstract

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.

Base Flipping within the α-Hemolysin Latch Allows Single-Molecule Identification of Mismatches in DNA

“…27,40 Fraying, which results in the localized opening of dsDNA structure at the termini of hairpins and duplexes has been widely reported, 41–44 and it is likely that opening of the duplex at the terminus is a prerequisite and/or plays a key role in nucleobase interactions with amino acids within αHL in these experiments. 27,40 …”

“…The unusual modulation in current signatures that we report here are similar to those described for DNA hairpin structures in a series of reports by Akeson and co-workers. 27–29,40 In their work, modulation between different residual current states during residence of a hairpin within the vestibule of αHL is attributed to interactions of the terminal base-pairs with the protein surface (probably lysine residues 25 ) near the 1.4 nm central constriction. 27–29,40 The nature of the interactions are highly dependent on the terminal base pair, with longer dwell times observed for some states when the terminal base pair is an AT as opposed to CG.…”

“…27–29,40 In their work, modulation between different residual current states during residence of a hairpin within the vestibule of αHL is attributed to interactions of the terminal base-pairs with the protein surface (probably lysine residues 25 ) near the 1.4 nm central constriction. 27–29,40 The nature of the interactions are highly dependent on the terminal base pair, with longer dwell times observed for some states when the terminal base pair is an AT as opposed to CG. 27,40 Fraying, which results in the localized opening of dsDNA structure at the termini of hairpins and duplexes has been widely reported, 41–44 and it is likely that opening of the duplex at the terminus is a prerequisite and/or plays a key role in nucleobase interactions with amino acids within αHL in these experiments.…”

“…27–29,40 The nature of the interactions are highly dependent on the terminal base pair, with longer dwell times observed for some states when the terminal base pair is an AT as opposed to CG. 27,40 Fraying, which results in the localized opening of dsDNA structure at the termini of hairpins and duplexes has been widely reported, 41–44 and it is likely that opening of the duplex at the terminus is a prerequisite and/or plays a key role in nucleobase interactions with amino acids within αHL in these experiments. 27,40 …”

Fabrication of charged nanopore channels using pulsed plasma deposition of methacrylic acid

“…The α-hemolysin protein ion channels are used for DNA, RNA and protein analysis [4][5][6][7][8]. Unfortunately, protein ion channels and lipid bilayers, used to suspend the protein channels, are not stable under variable temperatures, pH and salinity conditions [9]. These limitations have inspired the fabrication of solid-state nanopores.…”