() Extension of primers using the indicated diastereoisomers of dPTPαS with and 9°N (modified) DNA polymerases for the times indicated

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> The positions of migration of the unextended primer, primer extended by 1 nt and the primer extended by 2 nt, are indicated by P(N), N + 1, N + 2, respectively. The amount of oligonucleotide loaded to mark the position where the primer runs (lane 1) was less than for other lanes in the gel; () Exo III digestion of the products of 2-min primer extension reactions using the indicated diastereomers (S or R) of alpha-thio-dPTP (from a). Each primer extension product was purified with a QIAquick column and digested with 20 Units of Exo III for the indicated times. The control shows the degradation of 5′-P-labeled primer Z-SS-S19 (25mer) in a duplex with unlabeled Z-51-Temp, establishing that these are degraded by Exo III

One example of an ‘artifically expanded genetic information system’ (AEGIS)

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> Nucleobase pairing in this system conforms to the Watson–Crick geometry, with large purines (or purine analogs, both indicated by ‘pu’) pairing with small pyrimidines (or pyrimidine analogs, both indicated by ‘py’). The hydrogen-bonding acceptor (A) and donor (D) groups are listed from the major to the minor groove as indicated. The heterocycles shown are current implementations of the indicated hydrogen-bonding patterns; others are conceivable. Unshared pairs of electrons (or ‘electron density’) presented to the minor groove are shown by the shaded lobes. The nucleotides implementing the pyDDA:puAAD hydrogen-bonding pattern, the subject of this article, is at the bottom right

Exo III digestion of single- and double-stranded DNA containing phosphorothioate linkages

<p><b>Copyright information:</b></p><p>Taken from "Nucleoside alpha-thiotriphosphates, polymerases and the exonuclease III analysis of oligonucleotides containing phosphorothioate linkages"</p><p></p><p>Nucleic Acids Research 2007;35(9):3118-3127.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888802.</p><p>© 2007 The Author(s)</p> Denaturing (7 M urea) PAGE showing digestion of double-stranded substrate (the duplex between 5′-P-labeled G*-2S-51 or C*-2S-51 and the complementary C-51-Temp or G-51-Temp), and single-stranded substrate (5′-P-labeled G*-2S-51 or C*-2S-51). () Digestion with high concentrations of Exo III (0.5 or 2.5 U/µl, as indicated) for the indicated times. For dsDNA substrates, the ratio of G*-2S-51/C-51-Temp or C*-2S-51/G-51-Temp was 1/1 or 1/1.5 as indicated. () Digestion with low concentrations of Exo III (0.025 and 0.125 U/µl, as indicated) for the indicated times. The loading of the substrate 51mer was reduced to prevent overloading of the gel; thus, the absolute intensities of these bands cannot be compared with the intensities in other lanes in the gel

Synthesis and Properties of 5-Cyano-Substituted Nucleoside Analog with a Donor–Donor–Acceptor Hydrogen-Bonding Pattern

6-Aminopyridin-2-ones form Watson–Crick pairs with complementary purine analogues to add a third nucleobase pair to DNA and RNA, if an electron-withdrawing group at position 5 slows oxidation and epimerization. In previous work with a nucleoside analogue trivially named d<b>Z</b>, the electron withdrawing unit was a nitro group. Here, we describe an analogue of d<b>Z</b> (cyano-d<b>Z</b>) having a cyano group instead of a nitro group, including its synthesis, p<i>K</i>_a, rates of acid-catalyzed epimerization, and enzymatic incorporation

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

Pairwise distance estimates for orthologous intronic regions in a dataset composed of non-<i>ADH1</i> genes.

<p>Pairwise distance estimates for orthologous intronic regions in a dataset composed of non-<i>ADH1</i> genes.</p

Estimate of the ADH1 paralog duplications relative to the time of the major primate speciation events.

<p>The average pairwise distances separating the introns of the <i>ADH1</i> paralogs were compared with the average pairwise distances separating a set of introns in paired taxa. (A) This schematic illustrates the various ortholog comparisons used to estimate the relative age among the ADH1 paralogs. (B) This plot summaries the data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041175#pone-0041175-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041175#pone-0041175-t003" target="_blank">3</a>. The distances among the <i>ADH1</i> paralogs in marmoset, macaque and human (black diamonds) are somewhat larger than those separating catarrhine and platyrrhine orthologs (green circles), implying that these <i>ADH1</i> paralogs diverged (duplicated) before the catarrhine-platyrrhine split. Conversely, distances separating the <i>ADH1</i> paralogs in marmoset, macaque and human are somewhat smaller than those separating orthologous introns among strepsirhine and haplorhine (red squares), implying that these <i>ADH1</i> paralogs diverged after the split between strepsirhine and haplorhine.</p

Overview of primate phylogeny.

<p>An overview of primate phylogeny is shown, with the number of <i>ADH1</i> paralogs identified within select taxon indicated by the circled numbers at the leaves of the tree. Black numbers are derived from analysis of public databases, while red numbers were determined from cDNA sequencing reported here. The “4+1” designation for the macaque taxon indicates the presence of four <i>ADH1</i> paralogous genes plus one <i>ADH1</i> pseudogene. The genome sequencing projects are not completed for any lemur, so additional <i>ADH1</i> paralogs may be present (see text).</p