Nanopore Sequencing of an Expanded Genetic Alphabet Reveals High-Fidelity Replication of a Predominantly Hydrophobic Unnatural Base Pair

Unnatural
base pairs (UBPs) have been developed and used for a variety of in vitro applications as well as for the engineering of
semisynthetic organisms (SSOs) that store and retrieve increased information.
However, these applications are limited by the availability of methods
to rapidly and accurately determine the sequence of unnatural DNA.
Here we report the development and application of the MspA nanopore
to sequence DNA containing the dTPT3–dNaM UBP. Analysis of two sequence contexts reveals that DNA containing
the UBP is replicated with an efficiency and fidelity similar to that
of natural DNA and sufficient for use as the basis of an SSO that
produces proteins with noncanonical amino acids.