title: Synthesis and Enzymology of 2'-Deoxy-7-deazaisoguanosine Triphosphate and Its Complement: A Second Generation Pair in an Artificially Expanded Genetic Information System.
abstract: 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.
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States.
authors.forename: Nilesh B
authors.initials: NB
authors.lastname: Karalkar
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Leal
authors.affiliation: Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Myong-Sang
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bradley
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME) , 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States.|Firebird Biomolecular Sciences LLC , 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D060326:Chemistry Techniques, Synthetic; D003854:Deoxyribonucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D006860:Hydrogen Bonding; D016133:Polymerase Chain Reaction / Q000379:methods*; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods*; D019914:Taq Polymerase / Q000235:genetics / Q000378:metabolism
chemical_list: C000615615:2'-deoxy-7-deazaisoguanosine triphosphate; C113087:7-deazaisoguanine; D003854:Deoxyribonucleotides; D011743:Pyrimidines; D011758:Pyrroles; D019914:Taq Polymerase
keywords: 2′-deoxy-7-deazaisoguanosine-5′-triphosphate; PCR amplification; artificially expanded genetic information systems