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Dr. Jacek Cieślak Dr. Cristina Ausín Dr. Andrzej Grajkowski Dr. Serge L. Beaucage 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(14):4623-4632
The reaction of 2‐cyano‐2‐methyl propanal with 2′‐O‐aminooxymethylribonucleosides leads to stable and yet reversible 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl)ribonucleosides. Following N‐protection of the nucleobases, 5′‐dimethoxytritylation and 3′‐phosphitylation, the resulting 2′‐protected ribonucleoside phosphoramidite monomers are employed in the solid‐phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5‐benzylthio‐1H‐tetrazole, coupling efficiencies averaging 99 % are obtained within 180 s. Upon completion of the RNA‐chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) protective groups is effected (without releasing RNA alkylating side‐products) by treatment with tetra‐n‐butylammonium fluoride (0.5 m) in dry DMSO over a period of 24–48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2′‐O‐aminooxymethylribonucleosides in the design of new 2′‐hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost‐effective method for the chemical synthesis of high‐quality RNA sequences in the framework of RNA interference applications. 相似文献
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CraigC. Mello 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2007,119(37):7114-7124
Es ist wunderbar, heute hier zu sein. Beginnen möchte ich mit dem wichtigsten Part, nämlich Danke zu sagen. Zu allererst danke ich Andy Fire, der mir über all die Jahre ein großartiger Kollege und Freund war. Ohne Andy stünde ich heute nicht vor Ihnen. Dank schulde ich der University of Massachusetts, nicht nur für Ausrüstung, Laborräume und Geld, sondern vor allem auch für die großartigen Kollegen, mit denen zusammen ich meinen Forschungen nachgehe. Ohne die UMass und das tolle Umfeld dort, wäre ich wahrscheinlich heute nicht hier. Und nicht zu vergessen natürlich meine Familie; ich will jetzt keine Zeit aufbringen, jedem einzelnen zu danken, aber ihr wisst, wie wichtig ihr für mich seid. 相似文献
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High‐Resolution N6‐Methyladenosine (m6A) Map Using Photo‐Crosslinking‐Assisted m6A Sequencing
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Kai Chen Zhike Lu Xiao Wang Dr. Ye Fu Dr. Guan‐Zheng Luo Nian Liu Dr. Dali Han Dr. Dan Dominissini Dr. Qing Dai Prof. Tao Pan Prof. Chuan He 《Angewandte Chemie (International ed. in English)》2015,54(5):1587-1590
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Lukas Rigger Rachel L. Schmidt Kaitlyn M. Holman Prof. Miljan Simonović Prof. Dr. Ronald Micura 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(47):15872-15878
The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNASec) in all domains of life. The multistep pathway involves O‐phosphoseryl‐tRNA:selenocysteinyl‐tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNASec intermediate is converted into selenocysteinyl‐tRNASec. The SepSecS architecture and the mode of tRNASec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNASec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl‐derived tRNASec mimics that contain a hydrolysis‐resistant ribose 3′‐amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site‐specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)‐2‐amino‐4‐phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three‐strand enzymatic ligation protocol to obtain the corresponding full‐length tRNASec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS‐tRNA interactions. The novel tRNASec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X‐ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA‐dependent enzymes. 相似文献
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