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首页> 外文期刊>Journal of Systems Chemistry >Enzymatic synthesis of DNA employing pyrophosphate-linked dinucleotide substrates
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Enzymatic synthesis of DNA employing pyrophosphate-linked dinucleotide substrates

机译：使用焦磷酸盐连接的二核苷酸底物酶促合成DNA

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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.
机译：背景技术在理解自然界信息系统的起源中剩下的问题之一是合成第一批核酸的方式。使用核苷一磷酸的核苷三磷酸或咪唑啉化物作为构件可以实现这一点。备选地，二核苷焦磷酸盐可以用于该目的。使用由焦磷酸盐连接的二核苷酸组成的构件的优势可能是，在没有产物抑制的情况下，初始信息（二核苷酸）的指数增长成为可能。结果在本文中，我们证明了焦磷酸二核苷能够充当HIV-1 RT和几种热稳定DNA聚合酶的底物。在单掺入法中，化合物dAppdA能够通过Therminator DNA聚合酶以100μM以上的底物浓度100％转化为（P + 1）链。通过链延伸实验获得了全长延伸，Taq和Vent（exo -）DNA聚合酶的（P + 7）产物收率超过95％ 。有趣的是，使用异二聚体dAppdT可以将二核苷酸底物的任一核苷酸成分添加到DNA链中，这由模板程序定义。结论这项研究表明，焦磷酸二核苷可以被认为是模板指导的DNA合成中聚合酶的新型底物。从理论上讲，使用异二聚体作为底物，可以使用两个构件（dAppdT和dGppdC）而不是四个构件来酶促合成DNA。鉴于核苷酸掺入的Km值很差，聚合酶的进化对于使该方法实际应用将变得十分必要。

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