Enzymatic synthesis of DNA employing pyrophosphate-linked dinucleotide substrates

Herein, we demonstrate that dinucleoside pyrophosphates are able to act as substrate for HIV-1 RT and several thermostable DNA polymerases. In single incorporation assay, compound dAppdA was able to give a 100% conversion to the (P+1) strand by Therminator DNA polymerase and at a substrate concentration above 100 μM. Full-length elongation was obtained in a chain elongation experiment, with over 95% yield of (P+7) product by Taq and Vent (exo-) DNA polymerase. Interestingly, using heterodimer dAppdT addition of either nucleotide component of the dinucleotide substrate into the DNA chain can occur, which is defined by the template program.This study shows that dinucleoside pyrophosphates can be considered as a new type of substrate for polymerases in the template-directed DNA synthesis. Using heterodimers as substrate, theoretically, it is possible to synthesize DNA enzymatically using two building blocks (dAppdT and dGppdC) instead of four. Given the poor Km value for the nucleotide incorporation, evolution of polymerases will become necessary to make this process of practical use.The phosphorylation of nucleosides and nucleoside monophosphates under prebiotic conditions and leading to the formation of nucleoside diphosphates and nucleoside triphosphates was studied in the seventies of the previous century [1-5]. These studies were performed based on the observation that four nucleoside triphosphates are used as building blocks for the synthesis of each natural information system (DNA and RNA). In the context of the question "why this and not that", it is not unreasonable to ask the question why Nature uses four reagents to synthesize information systems and not two, composed of, for example, dinucleoside pyrophosphates. Dinucleoside pyrophosphates are not alien to cellular biology, demonstrated by the coenzyme function of NAD+ (Figure 1) and NADP+ in redox reactions. Moreover, NAD+ is used by poly(ADP-ribose) polymerase to synthesize PAR (Poly ADP ribose) [6] polym