Electrical Current Signatures of DNA Base Modifications in Single Molecules Immobilized in the α-Hemolysin Ion...

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.