Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages

The use of DNA polymerases to incorporate phosphorothioate linkages into DNA, and the use of exonuclease III to determine where those linkages have been incorporated, are re-examined in this work. The results presented here show that exonuclease III degrades single-stranded DNA as a substrate and digests through phosphorothioate linkages having one absolute stereochemistry, assigned (assuming inversion in the polymerase reaction) as S, but not the other absolute stereochemistry. This contrasts with a general view in the literature that exonuclease III favors double-stranded nucleic acid as a substrate and stops completely at phosphorothioate linkages. Furthermore, not all DNA polymerases appear to accept exclusively the (R) stereoisomer of nucleoside alpha-thiotriphosphates [and not the (S) diastereomer], a conclusion inferred two decades ago by examination of five Family-A polymerases and a reverse transcriptase. This suggests that caution is appropriate when extrapolating the detailed behavior of one polymerase from the behaviors of other polymerases. Furthermore, these results provide constraints on how exonuclease III–thiotriphosphate–polymerase combinations can be used to analyze the behavior of the components of a synthetic biology.