Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

Abstract

The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2'-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5' or 3' directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

Figure 1

Benzo[a]pyrene metabolism leading to guanine adducts in DNA.

Figure 2

Proposed model for translocation of a 4-mer and 4-mer BPDE adduct through the αHL nanopore. (A) Representative i-t trace for the 4-mer (5′-CCGC-3′) strand, (B) representative i-t trace for a 4-mer BPDE adducted oligomer. All data were recorded at 180 mV (trans vs. cis) in 1 M KCl at 25.0 ± 0.5°C with a 100 kHz low-pass filter and 500 kHz data acquisition rate.

Figure 3

Event types detected during translocation of the 41-mer BPDE sample. (A) Representative i-t traces for translocation of the 41-mer BPDE sample, (B) blowup of a 3′-entry event, and (C) blowup of a 5′-entry event. The data were recorded at 180 mV (trans vs. cis) at 25.0 ± 0.5°C. The data were refiltered to 50 kHz. Results from measurements are presented as percent ratio of the blockage current vs. open channel current %(I/I_o). The i t traces for events >50 μs were analyzed. Long open channel current segments (20 - 500 ms) were manually removed, as indicated on the i-t trace. A relatively low capture rate (~70 events/s) was observed due to the low concentration (2 μM) of the 41-mer BPDE studied.(D) Proposed model for the translocation of a 41-mer BPDE adduct through αHL. (I) DNA enters from the cis side of the channel by threading either the 3′ or 5′ tail. (II) The BPDE adduct becomes caught at the 1.4 nm central constriction that gives rise to the deep blockage in the ion current recorded that marks the presence of the BPDE adduct. (III)The DNA translocates through the β-barrel.

Figure 4

Current histograms for the step-current levels monitored for the 41-mer BPDE events. (A) Plots of frequency distributions for the I₂ and I_2′ current levels. (B) Plots of frequency distributions for the I₃ current levels. The data were collected at 120, 160 and 180 mV (trans vs. cis) in 1 M KCl at 25.0 ± 0.5°C and plotted with a bin size of 0.5 pA. Population distributions represent 400–450 events.