We present cognate base pair selectivity in template‐dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds‐Pa. T4 DNA ligase efficiently recognizes the Ds‐Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base‐pair selectivity, relative to the hydrogen‐bond interactions between pairing bases. The efficient ligation using Ds‐Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi‐synthetic organisms containing UBPs.

Genetic alphabet expansion technology, creating new replicable and functional DNA molecules with unnatural base pairs (UBPs), is the novel promising research area of xenobiology. Recently, this technology has rapidly advanced, resulting in the need for a sequencing method for DNA molecules containing UBPs. However, all of the conventional sequencing methods, such as Sanger methods, are for four‐letter DNA molecules. Here, we present an improved Sanger sequencing method (Sanger gap sequencing) for DNAs containing our UBP, Ds‐Px, which appears as gaps in the sequencing peak patterns. By improving the sequencing reaction for efficient Ds‐Px pairing and using modified Px substrates, we have developed a sequencing method with increased processivity and clear gap patterns for multiple Ds‐Px pairs in various sequence contexts. This method is useful for UBP applications such as high‐affinity DNA aptamer generation and semisynthetic organism creation involving UBPs. In addition, through this research, we found that the side chains of UBs greatly affect the efficiency of UB pairings in replication, thus suggesting further development of UBPs.