Polymerase-Mediated Incorporation of Unnatural Alkynylated Purine–Pyridazine Base Pairs

Wenjue Fan; Hidenori Okamura; Zhuoxin Dong; Giang Hoang Trinh; Yosuke Taniguchi; Fumi Nagatsugi

doi:10.1248/cpb.c26-00263

Regular Article

Polymerase-Mediated Incorporation of Unnatural Alkynylated Purine–Pyridazine Base Pairs

Wenjue Fan, Hidenori Okamura , Zhuoxin Dong, Giang Hoang Trinh, Yosuke Taniguchi, Fumi Nagatsugi

Author information

Wenjue Fan
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
Department of Chemistry, Graduate School of Science, Tohoku University, 6–3 Aramaki Aza-Aoba, Aoba-ku, Sendai 980–8578, Japan
Hidenori Okamura Corresponding author
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
Department of Chemistry, Graduate School of Science, Tohoku University, 6–3 Aramaki Aza-Aoba, Aoba-ku, Sendai 980–8578, Japan
Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1–1–1 Tsushima-naka, Kita-ku, Okayama 700–8530, Japan
Zhuoxin Dong
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
Department of Chemistry, Graduate School of Science, Tohoku University, 6–3 Aramaki Aza-Aoba, Aoba-ku, Sendai 980–8578, Japan
Giang Hoang Trinh
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
Department of Chemistry, Graduate School of Science, Tohoku University, 6–3 Aramaki Aza-Aoba, Aoba-ku, Sendai 980–8578, Japan
Yosuke Taniguchi
Faculty of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1–1–1 Tsushima-naka, Kita-ku, Okayama 700–8530, Japan
Fumi Nagatsugi Corresponding author
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2–1–1 Katahira, Aoba-ku, Sendai 980–8577, Japan
Department of Chemistry, Graduate School of Science, Tohoku University, 6–3 Aramaki Aza-Aoba, Aoba-ku, Sendai 980–8578, Japan

Keywords: unnatural base pair, nucleotide, DNA, polymerase reaction

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Supplementary material

2026 Volume 74 Issue 7 Pages 564-571

DOI https://doi.org/10.1248/cpb.c26-00263

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Abstract

Unnatural base pairs (UBPs) provide a promising strategy for expanding the genetic alphabet; however, their practical application requires efficient recognition by DNA polymerases. Here, we report the polymerase-mediated incorporation of alkynylated purine–pyridazine (Pu–Pz) base pairs bearing pseudo-nucleobase recognition units positioned in the major groove. Single-nucleotide insertion assays revealed that complementary Pu–Pz pairs are selectively incorporated into DNA by KOD DNA polymerase with efficiencies comparable to those of natural base pairs. Furthermore, efficient primer extension was maintained following UBP incorporation, indicating that bulky major-groove modifications do not significantly impede polymerase processivity. These results demonstrate the polymerase compatibility of major-groove-functionalized UBPs and highlight the potential of Pu–Pz pairs as building blocks for expanded genetic systems and site-specific DNA functionalization.

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