Synthesis and Enzymology of 2′-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System

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• 作者：Nilesh B. Karalkar ; Nicole A. Leal ; Myong-Sang Kim ; Kevin M. Bradley ; Steven A. Benner
• 刊名：ACS Synthetic Biology
• 出版年：2016
• 出版时间：July 15, 2016
• 年：2016
• 卷：5
• 期：7
• 页码：672-678
• 全文大小：352K
• 年卷期：0
• ISSN：2161-5063

文摘

As with natural nucleic acids, pairing between artificial nucleotides can be influenced by tautomerism, with different placements of protons on the heterocyclic nucleobase changing patterns of hydrogen bonding that determine replication fidelity. For example, the major tautomer of isoguanine presents a hydrogen bonding donor–donor–acceptor pattern complementary to the acceptor–acceptor–donor pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond donor–acceptor–donor pattern complementary to thymine. Calculations, crystallography, and physical organic experiments suggest that this tautomeric ambiguity might be “fixed” by replacing the N-7 nitrogen of isoguanine by a CH unit. To test this hypothesis, we prepared the triphosphate of 2′-deoxy-7-deazaiso-guanosine and used it in PCR to estimate an effective tautomeric ratio “seen” by Taq DNA polymerase. With 7-deazaisoguanine, fidelity-per-round was ∼92%. The analogous PCR with isoguanine gave a lower fidelity-per-round of ∼86%. These results confirm the hypothesis with polymerases, and deepen our understanding of the role of minor groove hydrogen bonding and proton tautomerism in both natural and expanded genetic “alphabets”, major targets in synthetic biology.