A Single Deoxynucleoside Kinase Variant from Drosophila melanogaster Synthesizes Monophosphates of Nucleosides That Are Components of an Expanded Genetic System.

Deoxynucleoside kinase from D. melanogaster (DmdNK) 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 DmdNK 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 DmdNK Q81E creates the monophosphates from the AEGIS nucleosides dP, dZ, dX, and dK (respectively 2-amino-8-(1'-ß-d-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one, dP; 6-amino-3-(1'-ß-d-2'-deoxyribofuranosyl)-5-nitro-1H-pyridin-2-one, dZ; 8-(1'ß-d-2'-deoxy-ribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione, dX; and 2,4-diamino-5-(1'-ß-d-2'-deoxyribofuranosyl)-pyrimidine, dK). Using a coupled enzyme assay, in vitro kinetic parameters were obtained for three of these nucleosides (dP, dX, and dK; the UV absorbance of dZ made it impossible to get its precise kinetic parameters). Thus, DmdNK Q81E appears to be a suitable enzyme to catalyze the first step in the biosynthesis of AEGIS 2'-deoxynucleoside triphosphates in vitro and, perhaps, in vivo, in a cell able to manage plasmids containing AEGIS DNA.

Expanded Genetic Alphabets: Managing Nucleotides That Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural Nucleotides.

2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Neither K nor X can form a Watson-Crick pair with any natural nucleobase. Further, neither K nor X has an accessible tautomeric form or a protonated/deprotonated state that can form a Watson-Crick pair with any natural nucleobase. In vitro experiments show how DNA polymerase I from E. coli manages replication of DNA templates with one K:X pair, but fails with templates containing two adjacent K:X pairs. In analogous in vivo experiments, E. coli lacking dKTP/dXTP cannot rescue chloramphenicol resistance from a plasmid containing two adjacent K:X pairs. These studies identify bacteria able to serve as selection environments for engineering cells that replicate AEGIS pairs that lack forms that are Watson-Crick complementary to any natural nucleobase.

Crystal structures of deprotonated nucleobases from an expanded DNA alphabet.

Reported here is the crystal structure of a heterocycle that implements a donor-donor-acceptor hydrogen-bonding pattern, as found in the Z component [6-amino-5-nitropyridin-2(1H)-one] of an artificially expanded genetic information system (AEGIS). AEGIS is a new form of DNA from synthetic biology that has six replicable nucleotides, rather than the four found in natural DNA. Remarkably, Z crystallizes from water as a 1:1 complex of its neutral and deprotonated forms, and forms a `skinny' pyrimidine-pyrimidine pair in this structure. The pair resembles the known intercalated cytosine pair. The formation of the same pair in two different salts, namely poly[[aqua(µ6-2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ido)sodium]-6-amino-5-nitropyridin-2(1H)-one-water (1/1/1)], denoted Z-Sod, {[Na(C5H4N3O3)(H2O)]·C5H5N3O3·H2O}n, and ammonium 2-amino-6-oxo-3-nitro-1,6-dihydropyridin-1-ide-6-amino-5-nitropyridin-2(1H)-one-water (1/1/1), denoted Z-Am, NH4+·C5H4N3O3-·C5H5N3O3·H2O, under two different crystallization conditions suggests that the pair is especially stable. Implications of this structure for the use of this heterocycle in artificial DNA are discussed.

Alternative Watson-Crick Synthetic Genetic Systems.

In its "grand challenge" format in chemistry, "synthesis" as an activity sets out a goal that is substantially beyond current theoretical and technological capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discovery and paradigm changes in ways that analysis cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biology: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural history on Earth. The outcomes in technology include new diagnostic tools that have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.

Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary Implications.

In addition to completing the Watson-Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:standard wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.

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'-ß-D-2'-deoxyribofuranosyl)-2(1H)-pyridone and 2-amino-8-(1'-ß-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one. These assays provide new ways to detect the formation of unnatural deoxyribonucleoside triphosphates in vitro and to confirm their incorporation into DNA. Thus, these assays can be used with other unnatural nucleotides.

Autonomous assembly of synthetic oligonucleotides built from an expanded DNA alphabet. Total synthesis of a gene encoding kanamycin resistance.

BACKGROUND: Many synthetic biologists seek to increase the degree of autonomy in the assembly of long DNA (L-DNA) constructs from short synthetic DNA fragments, which are today quite inexpensive because of automated solid-phase synthesis. However, the low information density of DNA built from just four nucleotide "letters", the presence of strong (G:C) and weak (A:T) nucleobase pairs, the non-canonical folded structures that compete with Watson-Crick pairing, and other features intrinsic to natural DNA, generally prevent the autonomous assembly of short single-stranded oligonucleotides greater than a dozen or so. RESULTS: We describe a new strategy to autonomously assemble L-DNA constructs from fragments of synthetic single-stranded DNA. This strategy uses an artificially expanded genetic information system (AEGIS) that adds nucleotides to the four (G, A, C, and T) found in standard DNA by shuffling hydrogen-bonding units on the nucleobases, all while retaining the overall Watson-Crick base-pairing geometry. The added information density allows larger numbers of synthetic fragments to self-assemble without off-target hybridization, hairpin formation, and non-canonical folding interactions. The AEGIS pairs are then converted into standard pairs to produce a fully natural L-DNA product. Here, we report the autonomous assembly of a gene encoding kanamycin resistance using this strategy. Synthetic fragments were built from a six-letter alphabet having two AEGIS components, 5-methyl-2'-deoxyisocytidine and 2'-deoxyisoguanosine (respectively S and B), at their overlapping ends. Gaps in the overlapped assembly were then filled in using DNA polymerases, and the nicks were sealed by ligase. The S:B pairs in the ligated construct were then converted to T:A pairs during PCR amplification. When cloned into a plasmid, the product was shown to make Escherichia coli resistant to kanamycin. A parallel study that attempted to assemble similarly sized genes with optimally designed standard nucleotides lacking AEGIS components gave successful assemblies of up to 16 fragments, but generally failed when larger autonomous assemblies were attempted. CONCLUSION: AEGIS nucleotides, by increasing the information density of DNA, allow larger numbers of DNA fragments to autonomously self-assemble into large DNA constructs. This technology can therefore increase the size of DNA constructs that might be used in synthetic biology.

Components of essential oils extracted from leaves and shoots of abies species in Japan.

The essential oils extracted from the leaves and the shoots of five Abies species (Pinaceae) growing in Japan, i.e., A. firma, A. homolepis, A. veitchii, A. mariesii, and A. sachalinensis, were characterized by GC-FID and GC/MS analyses. The yields of the essential oils extracted from A. sachalinensis were the highest among them. A significant amount of α-pinene was contained in the essential oils of all the Abies species examined. In A. homolepis and A. veitchii, significant differences in the content of the essential oils were found depending on whether these were extracted from the leaves or from the shoots. Regarding the enantiomeric ratio of α-pinene, the (+)-enantiomer was predominant in the oil extracted from the leaves of A. firma, while (-)-α-pinene was present in higher amounts in the oils of A. veitchii (leaves and shoots), A. mariesii (leaves and shoots), and A. sachalinensis (shoots). The fact that there may be a quantitative and qualitative difference in the components of the essential oils extracted from the different parts of a plant was investigated by cluster analysis.

Anxiolytic effect and tissue distribution of inhaled Alpinia zerumbet essential oil in mice.

The use of essential oils is common throughout the world, and clarification of their detailed effects and pharmaceutical potencies is necessary. Additionally, detailed information regarding the pharmacokinetics of essential oils is needed. In this report, GC-MS analysis was used to observe the tissue distribution of the multiple components of Alpinia zerumbet (Pers.) B.L. Burtt. et Sm. Anxiety-related behavior was evaluated by the light and dark box test (LD), open field test (OF), and elevated plus maze test (EPM). GC-MS quantification of the major components of A. zerumbet essential oil (AZEO) (alpha-pinene, p-cymene, 1,8-cineole, and limonene) was almost identical using either the injection or headspace injection method. All the behavioral assessments indicated that inhalation of AZEO had a positive anxiolytic effect. This was especially evident in the EPM (time spent in the open arms), where anxiolytic effects were clearly observed (P < 0.05). Alpha-Pinene accumulated in the brain at almost the same rate as in the liver. However, the oil components chiefly accumulated in the kidney. Therefore, the essential oil component in the largest proportion will not necessarily be distributed to organs throughout the body in the same quantities and/or ratios. It is necessary to consider tissue distribution for investigating the effects of essential oil inhalation.

Composition and seasonal variation of the essential oil from Abies sachalinensis from Hokkaido, Japan.

The composition of the steam-distilled essential oil from the leaves of Abies sachalinensis (F. Schmidt) Mast. cultivated in Hokkaido (Japan) was studied by GC-MS. The seasonal variation in the main volatile constituents was also investigated. Analysis of the essential oil resulted in the identification of 21 compounds with monoterpenes comprising 99.9% of the total. alpha-Pinene was the most abundant compound, followed by camphene, bornyl acetate, limonene, beta-pinene and beta-phellandrene. The sesquiterpene content was low, and was mainly represented by beta-caryophyllene, beta-caryophyllene, and gamma-selinene. The essential oil from the leaves, collected at eight different collection periods over more than 5 years, showed significant differences in composition. Alpha-Pinene was the predominant constituent during the collection periods, with a few exceptions. The alpha-pinene content of the oil was abundant in April-June, and decreased in November-December. Levels of bornyl acetate showed the greatest increase in December, when the temperature was very low. The enantiomeric distribution of alpha-pinene was suggested to relate to seasonal transformation. (+)-alpha-Pinene showed a tendency to increase when total precipitation, average temperature, and total duration of sunshine were high.

Effects of the essential oil from leaves of Alpinia zerumbet on behavioral alterations in mice.

In phytotherapy, the essential oil from the leaves of Alpinia zerumbet (Alpinia speciosa K. Schum.) (EOAZ) is used for neuropsychiatric symptoms, such as depression, stress and anxiety, and chronic problems that are associated with reproductive hormone imbalances in women. The chemical composition of EOAZ was analyzed by GC/MS, and the EOAZ properties inducing behavioral alterations in mice were examined by behavioral observations (BO) and an elevated plus-maze task (EPM), widely used as a method for assessing anxiolytic-like behaviors. Five major compounds, p-cymene (28.0 +/- 5.0%), 1,8-cineole (17.9 +/- 4.2%), terpinen-4-ol (11.9 +/- 6.3%), limonene (6.3 +/- 2.2%), and camphor (5.2 +/- 2.1%) were identified by retention indices, mass spectra and comparison with standards. Inhalational administration of EOAZ (8.7 ppm) induced unique jumping behaviors in mice. To further investigate the behavioral regulatory mechanisms of EOAZ, we administered an intraperitoneal injection of either 10 mg/kg 5-HTP or 10 mg/kg fluoxetine prior to the EOAZ inhalations. By 5-HTP or fluoxetine pretreatments, the jumping frequencies were significantly decreased. In EPM, EOAZ (0.087 and 8.7 ppm) obviously showed the anxiolytic-like activity in mice.

Composition and seasonal variation of essential oil in Alpinia zerumbet from Okinawa Island.

The composition of steam-distilled essential oils from the leaves of Alpinia zerumbet (Pers.) B. L. Burtt. et R. M. Sm. cultivated in Okinawa Island was investigated by GC-MS analysis. The seasonal variation of the main volatile constituents was also investigated. Analysis of the essential oil identified 17 compounds. It showed the predominant presence of monoterpenic constituents, representing 95% of the essential oil. p-Cymene was the most abundant compound, followed by 1,8-cineole, terpinen-4-ol, alpha-pinene, beta-pinene and limonene. The amount of sesqiterpenic content of the essential oil was small, mostly represented by beta-caryophyllene and alpha-caryophyllene. One of the phenylpropanoid derivatives, methyl cinnamate, was also detected. The essential oils from the leaves collected at ten different collection periods for over 5 years showed significant differences in their compositions. p-Cymene, terpinen-4-ol and 1,8-cineole were the most predominant constituents over the periods with a few exceptions. The content rates of p-cymene were abundant in summer, still high in late autumn and early winter, and decreased in mid-winter and early spring. In contrast, the contents of terpinen-4-ol and 1,8-cineole were high in winter, but decreased around summer. On enantiomeric distribution, terpinen-4-ol and alpha-pinene were suggested to relate to seasonal transformation. (+)-Terpinen-4-ol had a tendency to decrease in high temperatures and huge precipitations. It was also suggested that (-)-alpha-pinene was more sensitive to climate change than (+)-alpha-pinene.

[Clinical study on temporal lobe epilepsy in childhood caused by temporal lobe space-occupying lesions].

We studied the clinicoelectrical and neuroimaging features of 11 patients with symptomatic temporal lobe epilepsy (TLE) caused by temporal lobe space occupying lesions (SOLs), and compared its characteristics with those of 19 mesial TLE (MTLE) patients. Brain MRI demonstrated SOLs in the mesiotemporal lobe in 9, and laterotemporal lobe in the remaining 2 patients. Ten of the 11 patients successfully underwent surgery, which revealed tumors in 7 and focal cortical dysplasia in 3 patients. Comparisons of the clinical features between those with SOTLE and MTLE showed that both conditions shared the same clinical seizure manifestations such as gastric uprising sensation or ictal fear and a favorable response to surgery. However, the patients with SOTLE had fewer febrile convulsion, and more frequent seizure recurrences as well as TLE EEG discharges and associations of the monophasic clinical course than those with MTLE. In addition, the MRI findings were characterized by unilateral hippocampal atrophy in MTLE and expanding or SOLs in the SOTLE group. Children with complex partial seizures of suspected temporal lobe origin should undergo extensive neuroimaging evaluation.