Cytosine (C)-rich DNA can adopt i-motif folds under acidic conditions, with the human telomere i-motif providing a well-studied example. The dimensions of this i-motif are appropriate for capture in the nanocavity of the α-hemolysin (α-HL) protein pore under an electrophoretic force. Interrogation of the current vs time (i–t) traces when the i-motif interacts with α-HL identified characteristic signals that were pH dependent. These features were evaluated from pH 5.0 to 7.2, a region surrounding the transition pH of the i-motif (6.1). When the i-motif without polynucleotide tails was studied at pH 5.0, the folded structure entered the nanocavity of α-HL from either the top or bottom face to yield characteristic current patterns. Addition of a 5′ 25-mer poly-2′-deoxyadensosine tail allowed capture of the i-motif from the unfolded terminus, and this was used to analyze the pH dependency of unfolding. At pH values below the transition point, only folded strands were observed, and when the pH was increased above the transition pH, the number of folded events decreased, while the unfolded events increased. At pH 6.8 and 7.2 4% and 2% of the strands were still folded, respectively. The lifetimes for the folded states at pH 6.8 and 7.2 were 21 and 9 ms, respectively, at 160 mV electrophoretic force. These lifetimes are sufficiently long to affect enzymes operating on DNA. Furthermore, these transient lifetimes are readily obtained using the α-HL nanopore, a feature that is not easily achievable by other methods.

Nanopore technology holds high potential for next-generation DNA sequencing. This method operates by drawing an individual single-stranded DNA molecule through a nanoscale pore while monitoring the current deflections that occur as the DNA passes through. Individual current levels for the four DNA nucleotides have been established by immobilization of an end biotinylated strand in the pore in which the nucleotide of interest is suspended at the most sensitive region of the ion channel. Due to the inherent reactivity of the DNA bases, many modified nucleotides in the genome exist resulting from oxidative and UV insults, among others. Herein, the current levels for the common DNA damages 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), uridine (U), abasic sites (AP), thymine dimers (T=T), thymine glycol (Tg) and 5-iodocytosine (5-I-C) were assessed via immobilization experiments. In some cases, the current difference between the damaged and canonical nucleotides was not well resolved; therefore, we took advantage of the chemical reactivity of the new functional groups present to make amine adducts that shifted the current levels outside the range of the native nucleotides. Among adducts studied, only the 2-aminomethyl-18-crown-6 adduct was able to give a large current shift in the immobilization experiment, as well as to be observed in a translocation experiment. The results show potential in providing current level modulators for identification of some types of DNA damage. In principle, any DNA base modification that can be converted chemically or enzymatically to an abasic site could be identified in this way.

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