Unnatural base pairs between 2- and 6-substituted purines and 2-oxo(1H)pyridine for expansion of the genetic alphabet.

An unnatural base pair between 2-amino-6-(2-thienyl)purine (denoted by s) and 2-oxo(1H)pyridine (denoted by y) shows high selectivity in transcription and translation. Toward the further development of unnatural base pairs that also have exclusive selectivity in replication, we examined the roles of the 2-amino and 6-thienyl groups of s using base pairs between y and purine-analogs, 6-thienylpurine and 2-amino-6-furanylpurine, as well as s. The results obtained from the thermal stability and DNA polymerase single-nucleotide insertion experiments suggest that the 2-amino group of s contributes toward the shape complementarity of the pairing with y, rather than the hydrogen bonding with the 2-keto group of y. In addition, the bulkiness of positions 2 and 6 of the unnatural purines cooperatively determines the selectivity of the noncanonical pairing with y or the natural pyrimidines in replication. This information is useful not only for the development of unnatural, orthogonal base pairs, but also for understanding the mechanisms of base pair formation in replication.