Structural destabilization of DNA duplexes containing single-base lesions investigated by nanopore measurements

Abstract

The influence of DNA duplex structural destabilization introduced by a single base-pair modification was investigated by nanopore measurements. A series of 11 modified base pairs were introduced into the context of an otherwise complementary DNA duplex formed by a 17-mer and a 65-mer such that the overhanging ends comprised poly(dT)23 tails, generating a representative set of duplexes that display a range of unzipping mechanistic behaviors and kinetic stabilities. The guanine oxidation products 8-oxo-7,8-dihydroguanine (OG), guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) were paired with either cytosine (C), adenine (A), or 2,6-diaminopurine (D) to form modified base pairs. The mechanism and kinetic rate constants of duplex dissociation were determined by threading either the 3' or 5' overhangs into an α-hemolysin (α-HL) channel under an electrical field and measuring the distributions of unzipping times at constant force. In order of decreasing thermodynamic stability (as measured by duplex melting points), the rate of duplex dissociation increases, and the mechanism evolves from a first-order reaction to two sequential first-order reactions. These measurements allow us to rank the kinetic stability of lesion-containing duplexes relative to the canonical G:C base pair in which the OG:C, Gh:C, and Sp:C base pairs are, respectively, 3-200 times less stable. The rate constants also depend on whether unzipping was initiated from the 3' versus 5' side of the duplex. The kinetic stability of these duplexes was interpreted in terms of the structural destabilization introduced by the single base-pair modification. Specifically, a large distortion of the duplex backbone introduced by the presence of the highly oxidized guanine products Sp and Gh leads to a rapid two-step unzipping. The number of hydrogen bonds in the modified base pair plays a lesser role in determining the kinetics of duplex dissociation.

Figure 1

Base pairing schemes for X:Y (X = G, OG, Sp or Gh; Y = C, A, or D). The base pairs G:A, G:C, OG:A and OG:C are drawn based on reported structural data, while the hydantoin base pairs are drawn based on predictions in refs. and . The duplexes are arranged in order of decreasing melting temperatures (from top down). The D-containing base pairs are predicted based on their H-bonding capabilities as discussed in the text. The melting temperatures are listed under each X:Y-containing duplex and have an average standard deviation of 0.6 °C (See Supporting Information for individual standard deviations in T_m).

Figure 2

Unzipping of a DNA duplex using an α-HL nanopore under an electrical field. (A) dsDNA strand dissociation within an α-HL. The red dot in the target strand defines the relative position of X (X = G, OG, Sp, or Gh) opposite to Y (Y = C, A, or D) in the duplex. The + and − signs show the polarity of the electrodes. (B) Example current-time trace (X:Y = OG:C) at an applied voltage of −120 mV (cis versus trans). The blockades that last up to hundreds of milliseconds correspond to unzipping events of duplexes (unzipping duration shown as t). Events with duration shorter than 0.5 ms correspond to translocation of excess 17-mer ssDNA (the 65-mer and 17-mer strands are present in a 1:5 mole ratio). The red and blue dashed lines show the current blockage levels associated with 5′ and 3′ entry, respectively.

Figure 3

Histograms of blockage current for the duplex formed by hybridizing the X-containing 65-mer with the Y-containing 17-mer at −120 mV. X:Y represents the modified base pair.

Figure 4

Histograms of unzipping time (t) via 3′ entry (left column) and 5′ entry (right column) at −120 mV for the duplex that contains X:Y (where X = G, OG, Gh or Sp, and Y = C, A, D). The unzipping time constants (τ) were obtained based on the fit (red or blue curves) of the t histograms using the corresponding kinetic model, either a first-order reaction (Type I, red) or two sequential first-order reactions (Type II, blue). If the strand dissociation follows the Type II model, the unzipping time constants for each step are presented without assignment of the first and second steps. The histograms are plotted on different time scales in order to emphasize the shape as a determinant for the model type.