The creation of unnatural base pairs (UBPs) has rapidly advanced the genetic alphabet expansion technology of DNA, requiring a new sequencing method for UB-containing DNAs with five or more letters. The hydrophobic UBP, Ds‒Px, exhibits high fidelity in PCR and has been applied to DNA aptamer generation involving Ds as a fifth base. Here, we present a sequencing method for Ds-containing DNAs, in which Ds bases are replaced with natural bases by PCR using intermediate UB substrates (replacement PCR) for conventional deep sequencing. The composition rates of the natural bases converted from Ds significantly varied, depending on the sequence contexts around Ds and two different intermediate substrates. Therefore, we made an encyclopaedia of the natural-base composition rates for all sequence contexts in each replacement PCR using different intermediate substrates. The Ds positions in DNAs can be determined by comparing the natural-base composition rates in both the actual and encyclopaedia data, at each position of the DNAs obtained by deep sequencing after replacement PCR. We demonstrated the sequence determination of DNA aptamers in the enriched Ds-containing DNA libraries isolated by aptamer generation procedures targeting proteins. This study also provides valuable information about the fidelity of the Ds‒Px pair in replication.
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This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore) and National Research Foundation of Singapore (NRF-CRP17-2017-07).