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

Assays To Detect the Formation of Triphosphates of Unnatural Nucleotides: Application to Escherichia coli Nucleoside Diphosphate Kinase

One frontier in synthetic biology seeks to move artificially expanded genetic information systems (AEGIS) into natural living cells and to arrange the metabolism of those cells to allow them to replicate plasmids built from these unnatural genetic systems. In addition to requiring polymerases that replicate AEGIS oligonucleotides, such cells require metabolic pathways that biosynthesize the triphosphates of AEGIS nucleosides, the substrates for those polymerases. Such pathways generally require nucleoside and nucleotide kinases to phosphorylate AEGIS nucleosides and nucleotides on the path to these triphosphates. Thus, constructing such pathways focuses on engineering natural nucleoside and nucleotide kinases, which often do not accept the unnatural AEGIS biosynthetic intermediates. This, in turn, requires assays that allow the enzyme engineer to follow the kinase reaction, assays that are easily confused by ATPase and other spurious activities that might arise through “site-directed damage” of the natural kinases being engineered. This article introduces three assays that can detect the formation of both natural and unnatural deoxyribonucleoside triphosphates, assessing their value as polymerase substrates at the same time as monitoring the progress of kinase engineering. Here, we focus on two complementary AEGIS nucleoside diphosphates, 6-amino-5-nitro-3-(1′-β-d-2′-deoxyribofuranosyl)-2­(1<i>H</i>)-pyridone and 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo­[1,2-<i>a</i>]-1,3,5-triazin-4­(8<i>H</i>)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates <i>in vitro</i> and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides

A Single Deoxynucleoside Kinase Variant from <i>Drosophila melanogaster</i> Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System

Deoxynucleoside kinase from <i>D. melanogaster</i> (<i>Dm</i>dNK) has broad specificity; although it catalyzes the phosphorylation of natural pyrimidine more efficiently than natural purine nucleosides, it accepts all four 2′-deoxynucleosides and many analogues, using ATP as a phosphate donor to give the corresponding deoxynucleoside monophosphates. Here, we show that replacing a single amino acid (glutamine 81 by glutamate) in <i>Dm</i>dNK creates a variant that also catalyzes the phosphorylation of nucleosides that form part of an artificially expanded genetic information system (AEGIS). By shuffling hydrogen bonding groups on the nucleobases, AEGIS adds potentially as many as four additional nucleobase pairs to the genetic “alphabet”. Specifically, we show that <i>Dm</i>dNK Q81E creates the monophosphates from the AEGIS nucleosides d<b>P</b>, d<b>Z</b>, d<b>X</b>, and d<b>K</b> (respectively 2-amino-8-(1′-β-d-2′-deoxyribofuranosyl)-imidazo­[1,2-<i>a</i>]-1,3,5-triazin-4­(8<i>H</i>)-one, d<b>P</b>; 6-amino-3-(1′-β-d-2′-deoxyribofuranosyl)-5-nitro-1<i>H</i>-pyridin-2-one, d<b>Z</b>; 8-(1′β-d-2′-deoxy-ribofuranosyl)­imidazo­[1,2-<i>a</i>]-1,3,5-triazine-2­(8<i>H</i>)-4­(3<i>H</i>)-dione, d<b>X</b>; and 2,4-diamino-5-(1′-β-d-2′-deoxyribofuranosyl)-pyrimidine, d<b>K</b>). Using a coupled enzyme assay, <i>in vitro</i> kinetic parameters were obtained for three of these nucleosides (d<b>P</b>, d<b>X</b>, and d<b>K</b>; the UV absorbance of d<b>Z</b> made it impossible to get its precise kinetic parameters). Thus, <i>Dm</i>dNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2′-deoxynucleoside triphosphates <i>in vitro</i> and, perhaps, <i>in vivo</i>, in a cell able to manage plasmids containing AEGIS DNA