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Gene Synthesis

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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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Multivalent N-Acetylgalactosamine-Conjugated siRNA Localizes in Hepatocytes and Elicits Robust RNAi-Mediated Gene Silencing.

J Am Chem Soc. 2014 Dec 1;

Authors: Nair JK, Willoughby JL, Chan A, Charisse K, Alam MR, Wang Q, Hoekstra M, Kandasamy P, Kel’in AV, Milstein S, Taneja N, O’Shea J, Shaikh S, Zhang L, van der Sluis RJ, Jung ME, Akinc A, Hutabarat R, Kuchimanchi S, Fitzgerald K, Zimmermann T, van Berkel TJ, Maier MA, Rajeev KG, Manoharan M

Abstract
Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

PMID: 25434769 [PubMed – as supplied by publisher]

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Synthesis, Dynamic Combinatorial Chemistry, and PCR Amplification of 3′-5′ and 3′-6′ Disulfide-linked Oligonucleotides.
Angew Chem Int Ed Engl. 2014 Nov 20;
Authors: Hansen DJ, Manuguerra I, Kjelstrup MB, Gothelf K…

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[Establishment of breast cancer MDA-MB-231 cell line stably over-expressing human TOX high mobility group box family member 3].

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 Nov;30(11):1154-8

Authors: Han C, Yue L, Yang Y, Jian B, Ma L, Liu J

Abstract
Objective To construct the lentiviral expression vector of human TOX high mobility group box family member 3 (TOX3) gene and the MDA-MB-231 cell line which stably over-expresses TOX3 gene. Methods TOX3 gene was synthesized by the gene synthesis method and amplified by PCR, and then cloned into pLVEF-1a/GFP-Puro vector to construct pLVEF-1a/GFP-Puro-TOX3 lentiviral vector. After restriction enzyme analysis and sequence identification, the lentiviral vector was packaged and the titer was detected. The human breast cancer MDA-MB-231 cells were transfected with the recombinant lentiviral vector and cultured selectively by puromycin to acquire stably transfected cells. MDA-MB-231 cells which expressed GFP were observed by fluorescence microcopy. And the expression levels of TOX3 mRNA and protein in transfected MDA-MB-231 cells were detected by real-time quantitative PCR(qRT-PCR) and Western blotting, respectively. Results Restriction enzyme digestion and sequence analysis demonstrated that the lentiviral expression vectors of pLVEF-1a/GFP-Puro and pLVEF-1a/GFP-Puro-TOX3 were successfully constructed, and the viral titers were respectively 2×10(8) TU/mL and 1×10(8) TU/mL after lentiviral packaging. And after being transfected, more than 95% cells expressed GFP under a fluorescence microscope. The results of qRT-PCR and Western blotting showed that, when compared with the MDA-MB-231-NC negative control group, the expression of TOX3 mRNA and protein significantly increased in the MDA-MB-231-TOX3 group. Conclusion The study successfully constructed lentiviral expression vector of TOX3 gene and obtained MDA-MB-231 cell line stably over-expressing TOX3 gene by transfection with the recombinant vector.

PMID: 25374079 [PubMed – in process]

Continue reading about [Establishment of breast cancer MDA-MB-231 cell line stably over-expressing human TOX high mobility group box family member 3].

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Independent Generation and Reactivity of Uridin-2′-yl Radical.

J Org Chem. 2014 Oct 17;

Authors: Paul R, Greenberg MM

Abstract
The uridin-2′-yl radical (1) has been proposed as an intermediate during RNA oxidation. However, its reactivity has not been thoroughly studied due to the complex conditions under which it is typically generated. The uridin-2′-yl radical was independently generated from a benzyl ketone (2a) via Norrish type I photocleavage upon irradiation at λmax = 350 nm. Dioxygen and β-mercaptoethanol are unable to compete with loss of uracil from 1 in phosphate buffer. Thiol trapping competes with uracil fragmentation in less polar solvent conditions. This is ascribed mostly to a reduction in the rate constant for uracil elimination in the less polar solvent. Hydrogen atom transfer to 1 from β-mercaptoethanol occurs exclusively from the α-face to produce arabinouridine. Mass balances range from 72 to 95%. Furthermore, the synthesis of 2a is amenable to formation of the requisite phosphoramidite for solid-phase oligonucleotide synthesis. This and the fidelity with which the urdin-2′-yl radical is generated from 2a suggest that this precursor should be useful for studying the radical’s reactivity in synthetic oligonucleotides.

PMID: 25325847 [PubMed – as supplied by publisher]

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5-Nitroindole oligonucleotides with alkynyl side chains: universal base pairing, triple bond hydration and properties of pyrene “click” adducts.
Org Biomol Chem. 2014 Sep 19;
Authors: Ingale SA, Leo…

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Characterizing aptamer small molecule interactions with backscattering interferometry.
Analyst. 2014 Sep 17;
Authors: Kammer MN, Olmsted IR, Kussrow AK, Morris MJ, Jackson GW, Bornhop DJ
Abs…

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Synthesis of branched DNA using oxidatively cleavable tritylsulfenyl as a hydroxy protecting group.

Curr Protoc Nucleic Acid Chem. 2014;58:2.18.1-2.18.19

Authors: Seio K, Kanamori T, Sekine M

Abstract
The application of oxidatively cleavable tritylsulfenyl (TrS) group to the synthesis of branched DNA is described. The TrS protecting group can be removed by treatment with 1 M aqueous iodine, while it is stable toward an oxaziridine-type oxidant. At the same time, the sulfur-oxygen linkage showed sufficient stability under the acidic and basic conditions used in oligonucleotide synthesis. These properties of the TrS group enabled the synthesis of branched DNA using a branched phosphoramidite in which the two hydroxy groups are protected by a 4,4′-dimethoxytrityl (DMTr) group or a TrS group. In this unit, we describe an example of the synthesis of a three-way branched DNA using a branched phosphoramidite. Curr. Protoc. Nucleic Acid Chem. 58:2.18.1-2.18.19. © 2014 by John Wiley & Sons, Inc.

PMID: 25199636 [PubMed – in process]

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admin on September 10th, 2014
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Screening of accurate clones for gene synthesis in yeast.

J Biosci Bioeng. 2014 Sep 5;

Authors: Yarimizu T, Nakamura M, Hoshida H, Akada R

Abstract
Methods for error-less gene synthesis are desired because synthesized genes often contain mutations. By cloning PCR-assembled oligonucleotide fragments fused to a selection marker in yeast, we developed a novel method to screen accurate clones in gene synthesis. As a model case, the 555-bp luciferase gene from Gaussia princeps (GLuc) was synthesized to contain yeast-optimized codons (called yGLuc hereafter). After standard PCR-mediated oligonucleotide assembly, many clones showed no luciferase activity. Of these clones, most contained randomly located nucleotide deletions that produced frameshifts and resulted in premature termination. To exclude clones with premature termination, the synthesized yGLuc gene was cloned in-frame to fuse with the URA3 coding sequence, which served as a selection marker in the yeast Kluyveromyces marxianus. Ura(+) transformation selection was expected to eliminate clones with frameshift errors. The results showed that in-frame marker selection increased the frequency of active yGLuc gene to 79%. We used this strategy to synthesize the 1812-bp gene from Rhizopus oryzae that encodes glucoamylase. Five out of seven Ura(+) clones exhibited amylase activity. Of the functional clones, one contained the correct sequence, and four contained sequences with nucleotide changes, suggesting that in-frame selection frequently produced functional mutants. The K. marxianus non-homologous end joining mediated cloning method for gene synthesis will be useful for synthetic biological studies.

PMID: 25201012 [PubMed – as supplied by publisher]

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