Enzymatic synthesis of DNA employing pyrophosphate-linked dinucleotide substrates

Abstract

Abstract Background One of the remaining questions in the understanding of the origin of Nature's information system is the way the first nucleic acids have been synthesized. This could have been realized using nucleoside triphosphates or imidazolides of nucleoside monophosphates as building blocks. Alternatively, dinucleoside pyrophosphates could have been used for this purpose. The advantage of using building blocks, composed of pyrophosphate-linked dinucleotides, could be that exponential growth of initial information (dinucleotides) without product inhibition might become possible. Results 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. Conclusions 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.