Degenerate azole nucleosides direct the incorporation of mixtures of nucleotides in reactions catalyzed by thermostable DNA polymerases

DNA that encodes elements for degenerate replication events by use of artificial nucleobases offers a versatile approach to manipulating sequences for applications in biotechnology. A series of azole-3-carboxamide and 3-nitroazole deoxyribonucleosides have been designed as artificial nucleobases with the potential to exhibit multiple hydrogen bonding orientations within a single structural unit arising from internal bond rotations. These compounds were evaluated for their potential to influence degenerate nucleotide incorporation by thermostable DNA polymerases. Analyses included the incorporation of these analogues into a single position of a PCR primer followed by DNA amplification and sequencing of the PCR products. These nucleobase surrogates were able to influence the incorporation of the four natural deoxyribonucleotides over a range of specificity from minimal to high. Subtle alteration in the electronic structure of these template azole-nucleobases influenced the recognition by DNA polymerase, thus dictating substrate selection. These results significantly extend the molecular landscape for the design of biochemically functional nucleic acid analogues.