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.