Faithful PCR Amplification of an Unnatural Base‐Pair Analogue with Four Hydrogen Bonds

In vitro replication of an unnatural imidazopyridopyridine:naphthyridine base pair, (i.e., ImN N :NaO O ), having four hydrogen bonds was investigated. Kinetic studies of single‐nucleotide insertion revealed that ImN N and NaO O were recognized as complementary bases by an exonuclease‐deficient Klenow fragment with higher specificity and efficiency than two previously described pairs (ImN O :NaO N and ImO N :NaN O ) because of higher thermal and thermodynamic stabilities and the DAAD:ADDA (D=donor, A=acceptor) hydrogen‐bonding pattern of the ImN N :NaO O pair. Faithful polymerase chain reaction (PCR) amplification of a DNA fragment containing the ImN N :NaO O pair was achieved by using DNA polymerases possessing 3′→5′ exonuclease activity (≈99.5 % per doubling).