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Gene Synthesis

A method for multiplex gene synthesis employing error correction based on expression.
PLoS One. 2015;10(3):e0119927
Authors: Hsiau TH, Sukovich D, Elms P, Prince RN, Stritmatter T, Ruan P, Curry B, Anderson P, Sam…

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Active site and laminarin binding in glycoside hydrolase family 55.

J Biol Chem. 2015 Mar 9;

Authors: Bianchetti CM, Takasuka TE, Deutsch S, Udell HS, Yik EJ, Bergeman LF, Fox BG

Abstract
The Carbohydrate Active Enzyme (CaZY) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β -1,3-glucanases. The founding structure of the GH55 is PcLam55A from the white-rot fungus Phanaerochaete chrysosporium (Ishida, T., et al. (2009) J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies implicate Glu502 as the catalytic acid (as proposed earlier for Glu663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ~30% of the GH55 family gave 34 active enzymes (19% functional coverage of the non-redundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities, and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate GH phylogenetic space for functional properties.

PMID: 25752603 [PubMed – as supplied by publisher]

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Duplex DNA and DNA-RNA Hybrids with Parallel Strand Orientation: 2′-Deoxy-2′-fluoroisocytidine, 2′-Deoxy-2′-fluoroisoguanosine, and Canonical Nucleosides with 2′-Fluoro Substituents Cause Unexpected Changes on the Double Helix Stability.

J Org Chem. 2015 Mar 5;

Authors: Ingale SA, Leonard P, Tran QN, Seela F

Abstract
Oligonucleotides with parallel or antiparallel strand orientation incorporating 2′-fluorinated 2′-deoxyribonucleosides with canonical nucleobases or 2′-deoxy-2′-fluoroisocytidine ((F)iCd, 1c) and 2′-deoxy-2′-fluoroisoguanosine ((F)iGd, 3c) were synthesized. To this end, the nucleosides 1c and 3c as well as the phosphoramidite building blocks 19 and 23 were prepared and employed in solid-phase oligonucleotide synthesis. Unexpectedly, (F)iCd is not stable during oligonucleotide deprotection (55 °C, aq NH3) and was converted to a cyclonucleoside (14). Side product formation was circumvented when oligonucleotides were deprotected under mild conditions (aq ammonia-EtOH, rt). Oligonucleotides containing 2′-fluoro substituents ((F)iCd, (F)iGd and fluorinated canonical 2′-deoxyribonucleosides) stabilize double-stranded DNA, RNA, and DNA-RNA hybrids with antiparallel strand orientation. Unexpected strong stability changes are observed for oligonucleotide duplexes with parallel chains. While fluorinated oligonucleotides form moderately stable parallel stranded duplexes with complementary DNA, they do not form stable hybrids with RNA. Furthermore, oligoribonucleotide duplexes with parallel strand orientation are extremely unstable. It is anticipated that nucleic acids with parallel chains might be too rigid to accept sugar residues in the N-conformation as observed for ribonucleosides or 2′-deoxy-2′-fluororibonucleosides. These observations might explain why nature has evolved the principle of antiparallel chain orientation and has not used the parallel chain alignment.

PMID: 25742047 [PubMed – as supplied by publisher]

Continue reading about Duplex DNA and DNA-RNA Hybrids with Parallel Strand Orientation: 2′-Deoxy-2′-fluoroisocytidine, 2′-Deoxy-2′-fluoroisoguanosine, and Canonical Nucleosides with 2′-Fluoro Substituents Cause Unexpected Changes on the Double Helix Stability.

Practical Synthesis of Cytidine-5-Carboxamide-Modified Nucleotide Reagents.
Nucleosides Nucleotides Nucleic Acids. 2015 Mar 4;34(3):180-198
Authors: Rohloff JC, Fowler C, Ream B, Carter JD, Wardle G, Fitzwater T

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Improved Lower Bounds of DNA Tags Based on a Modified Genetic Algorithm.
PLoS One. 2015;10(2):e0110640
Authors: Wang B, Wei X, Dong J, Zhang Q
Abstract
The well-known massively paral…

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Related Articles

Synthesis of triazole-nucleoside phosphoramidites and their use in solid-phase oligonucleotide synthesis.

Curr Protoc Nucleic Acid Chem. 2014;55:4.57.1-4.57.38

Authors: Peel BJ, Efthymiou TC, Desaulniers JP

Abstract
Triazole-backbone oligonucleotides are macromolecules that have one or more triazole units that are acting as a backbone mimic. Triazoles within the backbone have been used within oligonucleotides for a variety of applications. This unit describes the preparation and synthesis of two triazole-nucleoside phosphoramidites [uracil-triazole-uracil (UtU) and cytosine-triazole-uracil (CtU)] based on a PNA-like scaffold, and their incorporation within oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 55:4.57.1-4.57.38. © 2014 by John Wiley & Sons, Inc.

PMID: 25631534 [PubMed – in process]

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admin on February 18th, 2015

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How do bacteria tune translation efficiency?
Curr Opin Microbiol. 2015 Jan 27;24C:66-71
Authors: Li GW
Abstract
Bacterial proteins are translated with precisely determined rates to …

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DNASynth: A Computer Program for Assembly of Artificial Gene Parts in Decreasing Temperature.
Biomed Res Int. 2015;2015:413262
Authors: Nowak RM, Wojtowicz-Krawiec A, Plucienniczak A
Abstra…

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Anti-herpes simplex virus activity of polysaccharides from Eucheuma gelatinae.
World J Microbiol Biotechnol. 2015 Jan 21;
Authors: Jin F, Zhuo C, He Z, Wang H, Liu W, Zhang R, Wang Y
Abstra…

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Design and synthesis of nucleolipids as possible activated precursors for oligomer formation via intramolecular catalysis: stability study and supramolecular organization.

J Syst Chem. 2014;5:5

Authors: Gangadhara KL, Srivastava P, Rozenski J, Mattelaer H, Leen V, Dehaen W, Hofkens J, Lescrinier E, Herdewijn P

Abstract
BACKGROUND: Fatty acid vesicles are an important part of protocell models currently studied. As protocells can be considered as pre-biological precursors of cells, the models try to contribute to a better understanding of the (cellular) origin of life and emphasize on 2 major aspects: compartmentalization and replication. It has been demonstrated that lipid-based membranes are amenable to growth and division (shell replication). Furthermore compartmentalization creates a unique micro-environment in which biomolecules can accumulate and reactions can occur. Pioneering research by Sugawara, Deamer, Luisi, Szostak and Rasmussen gave more insight in obtaining autocatalytic, self-replicating vesicles capable of containing and reproducing nucleic acid sequences (core replication). Linking both core and shell replication is a challenging feat requiring thorough understanding of membrane dynamics and (auto)catalytic systems. A possible solution may lie in a class of compounds called nucleolipids, who combine a nucleoside, nucleotide or nucleobase with a lipophilic moiety. Early contributions by the group of Yanagawa mentions the prebiotic significance (as a primitive helical template) arising from the supramolecular organization of these compounds. Further contributions, exploring the supramolecular scope regarding phospoliponucleosides (e.g. 5′-dioleylphosphatidyl derivatives of adenosine, uridine and cytidine) can be accounted to Baglioni, Luisi and Berti. This emerging field of amphiphiles is being investigated for surface behavior, supramolecular assembly and even drug ability.
RESULTS: A series of α/β-hydroxy fatty acids and α-amino fatty acids, covalently bound to nucleoside-5′-monophosphates via a hydroxyl or amino group on the fatty acid was examined for spontaneous self-assembly in spherical aggregates and their stability towards intramolecular cleavage. Staining the resulting hydrophobic aggregates with BODIPY-dyes followed by fluorescent microscopy gave several distinct images of vesicles varying from small, isolated spheres to higher order aggregates and large, multimicrometer sized particles. Other observations include rod-like vesicle precursors. NMR was used to assess the stability of a representative sample of nucleolipids. 1D (31)P NMR revealed that β-hydroxy fatty acids containing nucleotides were pH-stable while the α-analogs are acid labile. Degradation products identified by [(1)H-(31)P] heteroTOCSY revealed that phosphoesters are cleaved between sugar and phosphate, while phosphoramidates are also cleaved at the lipid-phosphate bond. For the latter compounds, the ratio between both degradation pathways is influenced by the nucleobase moiety. However no oligomerization of nucleotides was observed; nor the formation of 3′-5′-cyclic nucleotides, possible intermediates for oligonucleotide synthesis.
CONCLUSIONS: The nucleolipids with a deoxyribose sugar moiety form small or large vesicles, rod-like structures, vesicle aggregates or large vesicles. Some of these aggregates can be considered as intermediate forms in vesicle formation or division. However, we could not observe nucleotide polymerization or cyclic nucleotide function of these nucleolipids, regardless of the sugar moiety that is investigated (deoxyribose, ribose, xylose). To unravel this observation, the chemical stability of the constructs was studied. While the nucleolipids containing β-hydroxy fatty acids are stable as well in base as in acid circumstances, others degraded in acidic conditions. Phosphoramidate nucleolipids hydrolyzed by P-N as well as P-O bond cleavage where the ratio between both pathways depends on the nucleobase. Diester constructs with an α-hydroxy stearic acid degraded exclusively by hydrolysis of the 5′-O-nucleoside ester bond. As the compounds are too stable and harsh conditions would destruct the material itself, more reactive species such as lipid imidazolates of nucleotides need to be synthesized to further analyze the potential polymerization process. Graphical AbstractVesicle information of a nucleolipid consisting of a nucleoside 5′-monophosphate and a α-hydroxy fatty acid.

PMID: 25558290 [PubMed – as supplied by publisher]

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