Ribonucleosides for an Artificially Expanded Genetic Information System

Rearranging hydrogen bonding groups adds nucleobases to an artificially expanded genetic information system (AEGIS), pairing orthogonally to standard nucleotides. We report here a large-scale synthesis of the AEGIS nucleotide carrying 2-amino-3-nitro­pyridin-6-one (trivially Z) via Heck coupling and a hydro­boration/oxidation sequence. RiboZ is more stable against epimerization than its 2′-deoxy­ribo analogue. Further, T7 RNA polymerase incorporates ZTP opposite its Watson–Crick complement, imidazo­[1,2-a]-1,3,5-triazin-4­(8<i>H</i>)­one (trivially P), laying grounds for using this “second-generation” AEGIS Z:P pair to add amino acids encoded by mRNA

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

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 <i>donor</i>–<i>donor</i>–<i>acceptor</i> pattern complementary to the <i>acceptor</i>–<i>acceptor</i>–<i>donor</i> pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond <i>donor</i>–<i>acceptor</i>–<i>donor</i> 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 <i>Taq</i> 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

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

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 <i>donor</i>–<i>donor</i>–<i>acceptor</i> pattern complementary to the <i>acceptor</i>–<i>acceptor</i>–<i>donor</i> pattern of 5-methylisocytosine. However, in its minor tautomer, isoguanine presents a hydrogen bond <i>donor</i>–<i>acceptor</i>–<i>donor</i> 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 <i>Taq</i> 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