Artificially expanded genetic information system: a new base pair with an alternative hydrogen bonding pattern

To support efforts to develop a ‘synthetic biology’ based on an artificially expanded genetic information system (AEGIS), we have developed a route to two components of a non-standard nucleobase pair, the pyrimidine analog 6-amino-5-nitro-3-(1′-β-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ) and its Watson–Crick complement, the purine analog 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one (dP). These implement the pyDDA:puAAD hydrogen bonding pattern (where ‘py’ indicates a pyrimidine analog and ‘pu’ indicates a purine analog, while A and D indicate the hydrogen bonding patterns of acceptor and donor groups presented to the complementary nucleobases, from the major to the minor groove). Also described is the synthesis of the triphosphates and protected phosphoramidites of these two nucleosides. We also describe the use of the protected phosphoramidites to synthesize DNA oligonucleotides containing these AEGIS components, verify the absence of epimerization of dZ in those oligonucleotides, and report some hybridization properties of the dZ:dP nucleobase pair, which is rather strong, and the ability of each to effectively discriminate against mismatches in short duplex DNA

Genomic Analysis of the Hydrocarbon-Producing, Cellulolytic, Endophytic Fungus Ascocoryne sarcoides

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system

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

() 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

PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1

As the next step towards generating a synthetic biology from artificial genetic information systems, we have examined variants of HIV reverse transcriptase (RT) for their ability to synthesize duplex DNA incorporating the non-standard base pair between 2,4-diaminopyrimidine (pyDAD), a pyrimidine presenting a hydrogen bond ‘donor–acceptor–donor’ pattern to the complementary base, and xanthine (puADA), a purine presenting a hydrogen bond ‘acceptor–donor–acceptor’ pattern. This base pair fits the Watson–Crick geometry, but is joined by a pattern of hydrogen bond donor and acceptor groups different from those joining the GC and AT pairs. A variant of HIV-RT where Tyr 188 is replaced by Leu, has emerged from experiments where HIV was challenged to grow in the presence of drugs targeted against the RT, such as L-697639, TIBO and nevirapine. These drugs bind at a site near, but not in, the active site. This variant accepts the pyDAD-puADA base pair significantly better than wild type HIV-RT, and we used this as a starting point. A second mutation, E478Q, was introduced into the Y188L variant, in the event that the residual nuclease activity observed is due to the RT, and not a contaminant. The doubly mutated RT incorporated the non-standard pair with sufficient fidelity that the variant could be used to amplify oligonucleotides containing pyDAD and puADA through several rounds of a polymerase chain reaction (PCR) without losing the non-standard base pair. This is the first time where DNA containing non-standard base pairs with alternative hydrogen bonding patterns has been amplified by a full PCR. This work also illustrates a research strategy that combines in clinico pre-evolution of proteins followed by rational design to obtain an enzyme that meets a particular technological specification