Deoxynucleoside Triphosphate Containing Pyridazin-3-one Aglycon as a Thymidine Triphosphate Substitute for Primer Extension and Chain Elongation by Klenow Fragments

Deoxynucleoside 5'-triphosphate was synthesized with 3-oxo-2H-pyridazin-6-y1 (Pz(O)) a uracil analogue lacking a 2-keto group as the nucleobase. Theoretical analyses and hybridization experiments indicated that Pz(O) recognizes adenine (A) for formation of a Watson-Crick base pair. Primer extension reactions using nucleoside S'-triphosphate and the Klenow fragment revealed that the synthetic nucleoside S'-triphosphate was incorporated into the 3' end of the primer through recognition of A in the template strand. Moreover, the 3' nucleotide residue harboring Pz(O) as the base was resistant to the 3'-exonuclease activity of Klenow fragment exo+. The primer bearing the Pz(O) base at the 3' end could function in subsequent chain elongation. These properties of Pz(O) were attributed to the presence of an endocyclic nitrogen atom at the position ortho to the glycosidic bond, which was presumed to form an H-bond with the amino acid residue of DNA polymerase for effective recognition of the 3' end of the primer for primer extension. These results provide a basis for designing new nucleobases by combining a nitrogen atom at the position ortho to the glycosidic bond and base-pairing sites for Watson-Crick hydrogen bonding.