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Gene Synthesis

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]

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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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Systematic targeted gene deletion using the gene-synthesis method in fission yeast.
J Microbiol Methods. 2014 Aug 19;
Authors: Nam M, Lee SJ, Han S, Kim D, Lee M, Kang EJ, Park HO, Lee AR, Lee S, Ki…

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Optimization and scale-up of oligonucleotide synthesis in packed bed reactors using computational fluid dynamics modeling.

Biotechnol Prog. 2014 Jul 31;

Authors: Wolfrum C, Josten A, Götz P

Abstract
A computational fluid dynamics (CFD) model for the analysis of oligonucleotide synthesis in packed bed reactors was developed and used to optimize the scale up of the process. The model includes reaction kinetics data obtained under well defined conditions comparable to the situation in the packed bed. The model was validated in terms of flow conditions and reaction kinetics by comparison with experimental data. Experimental validation and the following model parameter studies by simulation were performed on the basis of a column with 0.3 g oligonucleotide capacity. The scale-up studies based on CFD modelling were calculated on a 440 g scale (oligonucleotide capacity). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 2014.

PMID: 25105730 [PubMed – as supplied by publisher]

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