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

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.