Recognizing a single base in an individual DNA strand: a step toward DNA sequencing in nanopores

Abstract

Functional supramolecular chemistry at the single-molecule level. Single strands of DNA can be captured inside α-hemolysin transmembrane pore protein to form single-species α-HL·DNA pseudorotaxanes. This process can be used to identify a single adenine nucleotide at a specific location on a strand of DNA by the characteristic reductions in the α-HL ion conductance. This study suggests that α-HL-mediated single-molecule DNA sequencing might be fundamentally feasible.

Figure 1

a) Schematic representation of the single-species α– HL·DNA pseudorotaxane formation. By employing sequences with a long ss-DNA segment having a hairpin structure at one end, the SS-DNA can thread the pore under positive applied potentials and held stably in a pseudorotaxane configuration by the interactions of the DNA duplex segment at the pore entrance. The heptameric transmembrane pore structure is depicted in a cutaway side view. b) Ion conductance blockades caused by capture of poly d(A) and poly d(C) single strands (sequences 1 and 2, respectively). The typical events shown are from ion conductance traces recorded at +170 mV when the thread molecules (1 μM) were added to the cis chamber. Traces were recorded under symmetrical conditions (500 mM KCl, 5 mM MOPS pH 7.5), Bessel filtered at 5 kHz, and sampled at 200 μs.

Figure 2

The averaged residual currents for various α-HL·DNA pseudorotaxanes measured at +170 mV. The left panel depicts the percent residual currents measured for poly d(A) and poly d(C) threads in two topoisomeric α-HL·DNA pseudorotaxane configurations. Note that when the α-HL pore is threaded with a homopolymeric DNA segment either from the free 3’-terminus (1 or 2) or from 5’-terminus (3 or 4), similar characteristic A-type or C-type ion channel blockades are observed. The right panel depicts the observed residual currents for α-HL·DNA pseudorotaxanes prepared using four different ss-DNA block copolymer threads (5–6). The region of the ss-DNA segment recognized by the α-HL channel (10 to 20 nucleotides away from the edge of the hairpin segment) is marked by the gray rectangle. For clarity A-type and C-type residual currents are shown as black and gray bars, respectively.

Figure 3

Percent probability of the A-type (I_R < 27%) and the C-type (I_R > 27%) residual currents for single deoxyadenosine-substituted poly d(C) DNA strands captured inside the α-HL pore as a pseudorotaxane. Percent probability was calculated by the number of A-type or C-type events measured at +170 mV divided by the total number of events (n) recorded for a given strand (n = 11, 21, 22, 32, 22, and 14 for strands 1, 2, 9, 10, 11, 12, 13, respectively). The percent probability of A-type and C-type events for each strand are given as black and gray bars respectively.

Figure 4

Percent probability of the A-type and the C-type events for single deoxyadenosine-substituted poly d(C) DNA hairpins sequences having either 13- or 10-basepair stem duplexes (strands 14–15, and 16–18, respectively) measured at +170 mV (n = 43, 44, 69, 35, 66 for 14–18, respectively). For clarity the A-type (I_R < 27%) and C-type (I_R > 27%) events are shown as black and grey bars, respectively.