Crown ether-electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

Abstract

DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na(+) produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li(+), Na(+) or K(+)) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement.

Fig. 1.

Synthetic scheme for AP-18c6 adduct.

Fig. 2.

Immobilization studies of AP-18c6 with 1 M NaCl as electrolyte. (A) Three types of i -t traces presented by AP-18c6. (B) Histograms of current blockage levels of both 3′ and 5′ entries. Color code: red (type 1), blue (type 2a), and black (type 2b).

Fig. 3.

Translocation studies of AP-18c6 in homopolymeric strands with 3 M NaCl as the electrolyte. (A) Typical i -t trace of the control strand and definition of translocation duration (t_D). (B) Sample density plots of control and adducted strands (120 mV trans vs. cis). (C) Plot of t_max vs. voltage for the control strand. (D) Plot of τ vs. voltage for the mono and bis adducts.

Fig. 4.

Individual i-t traces of AP-18c6 in homopolymeric strands. (A) Sample i-t traces of 3′ entry for mono and bis adducts (120 mV trans vs. cis). (B) Sample i-t traces of 5′ entry for mono and bis adducts (120 mV trans vs. cis).

Fig. 5.

Translocation studies of AP-18c6 in heteropolymeric sequence with 3 M NaCl as electrolyte. (A) Heteropolymeric sequence (60 mer). (B) Sample density plots of control (X = C) and adducted strand (X = AP-18c6) (120 mV trans vs. cis). (C) Individual i-t traces of the adducted strand. Top: 3′ entry event. Bottom: 5′ entry event. (D, Left) Plot of t_max vs. voltage for the control strand. (Right) Plot of τ vs. voltage for adducted strand.

Fig. 6.

Electrolyte-dependent translocation studies of the mono adduct strand. (A) Density plots of various %K⁺ (in 1 M LiCl) conditions under 80 mV (trans vs. cis). (B) Complexation and dissociation of 18c6-Na⁺ (R = DNA) with the equilibrium constant K_s (43). (C) Plot of combined percentage event counts of populations B and C vs. %K⁺ (in 1 M LiCl), compared to calculated values.