Nanopore Sequencing of “Alien” DNA Bases

Recently, it was shown for the first time that Escherichia coli can efficiently propagate a genetic alphabet expanded from the four canonical DNA nucleotides to include the synthetic “alien” bases d5SICS and dNaM. Traditional sequencing platforms cannot detect these alien bases. We tested whether nanopore sequencing with the protein pore Mycobacterium smegmatis porin A (MspA) is sensitive to d5SICS and dNaM. In nanopore sequencing, a nanometer scale pore provides the only electrical connection between two electrolyte solutions. An applied voltage causes a current to flow through the pore. Negatively-charged single stranded DNA is drawn through the pore, causing a nucleotide specific reduction in the measured current. Thus-far, MspA is the only nanopore shown to demonstrate single-nucleotide sensitivity to standard nucleotides as well as methyl cytosine and hydroxymethyl cytosine. We use the φ29 DNA polymerase to regulate DNA motion to single-nucleotide steps. Here, we demonstrate the direct detection d5SICS and dNaM with MspA-enabled nanopore sequencing.