Dynamics of a DNA mismatch site held in confinement discriminate epigenetic modifications of cytosine

The identification and discrimination of four epigenetic modifications to cytosine in the proposed active demethylation cycle is demonstrated at the single-molecule level, without the need for chemical pretreatment or labeling. The wild-type protein nanopore α-hemolysin is used to capture individual DNA duplexes containing a single cytosine–cytosine mismatch. The mismatch is held at the latch constriction of α-hemolysin, which is used to monitor the kinetics of base-flipping at the mismatch site. Base-flipping and the subsequent interactions between the DNA and the protein are dramatically altered when one of the cytosine bases is replaced with methyl-, hydroxymethyl-, formyl-, or carboxylcytosine. As well as providing a route to single-molecule analysis of important epigenetic markers in DNA, our results provide important insights into how the introduction of biologically relevant, but poorly understood, modifications to cytosine affect the local conformational dynamics of a DNA duplex in a confined environment

Energetics of base flipping at a DNA mismatch site confined at the latch constriction of α-hemolysin

Unique, two-state modulating current signatures are observed when a cytosine-cytosine mismatch pair is confined at the 2.4 nm latch constriction of the [small alpha]-hemolysin ([small alpha]HL) nanopore. We have previously speculated that the modulation is due to base flipping at the mismatch site. Base flipping is a biologically significant mechanism in which a single base is rotated out of the DNA helical stack by 180[degree]. It is the mechanism by which enzymes are able to access bases for repair operations without disturbing the global structure of the helix. Here, temperature dependent ion channel recordings of individual double-stranded DNA duplexes inside [small alpha]-HL are used to derive thermodynamic ([capital Delta]H, [capital Delta]S) and kinetic (Ea) parameters for base flipping of a cytosine at an unstable cytosine-cytosine mismatch site. The measured activation energy for flipping a cytosine located at the latch of [small alpha]HL out of the helix (18 +/- 1 kcal mol-1) is comparable to that previously reported for base flipping at mismatch sites from NMR measurements and potential mean force calculations. We propose that the [small alpha]HL nanopore is a useful tool for measuring conformational changes in dsDNA at the single molecule level

Effect of the electric double layer on the activation energy of ion transport in conical nanopores

Measured apparent activation energies, EA, of ion transport (K+ and Cl–) in conical glass nanopores are reported as a function of applied voltage (−0.5 to 0.5 V), pore size (20–2000 nm), and electrolyte concentration (0.1–50 mM). EA values for transport within an electrically charged conical glass nanopore differ from the bulk values due to the voltage and temperature-dependent distribution of the ions within the double layer. Remarkably, nanopores that display ion current rectification also display a large decrease in EA under accumulation mode conditions (at applied negative voltages versus an external ground) and a large increase in EA under depletion mode conditions (at positive voltages). Finite element simulations based on the Poisson–Nernst–Planck model semiquantitatively predict the measured temperature-dependent conductivity and dependence of EA on applied voltage. The results highlight the relationships between the distribution of ions with the nanopore, ionic current, and EA and their dependencies on pore size, temperature, ion concentration, and applied voltage

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

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