使用Fpmp化学方法制备含4-硫尿苷的寡核糖核苷酸。利用350纳米辐射对RNA结合蛋白进行光交联。
Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.
作者信息
McGregor A, Rao M V, Duckworth G, Stockley P G, Connolly B A
机构信息
Department of Biochemistry, The University of Newcastle, Newcastle upon Tyne, UK.
出版信息
Nucleic Acids Res. 1996 Aug 15;24(16):3173-80. doi: 10.1093/nar/24.16.3173.
Abstract
The preparation of a 4-thiouridine phosphoramidite suitable for RNA synthesis and its subsequent incorporation into oligoribonucleotides is described. The thiol group is protected with a 2-cyanoethyl group and the 2'-OH with a 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl function. Thiouridine-containing oligoribonucleotides were used as 350 nm UV crosslinking probes for the photoaffinity labelling of RNA binding proteins. Specific crosslinking was demonstrated between the Rev protein of HIV-1 (as a glutathione S-transferase fusion protein) and its RNA target, the Rev-responsive element. It was not possible to generate crosslinks between the RNA bacteriophage MS2 coat protein and the initiator stem-loop of the replicase gene, to which it binds. These results are consistent with the structural data available on both systems.
摘要
描述了一种适用于RNA合成的4-硫代尿苷亚磷酰胺的制备及其随后掺入寡核糖核苷酸的过程。硫醇基团用2-氰基乙基保护，2'-羟基用1-(2-氟苯基)-4-甲氧基哌啶-4-基官能团保护。含硫代尿苷的寡核糖核苷酸用作350nm紫外线交联探针，用于RNA结合蛋白的光亲和标记。已证明HIV-1的Rev蛋白（作为谷胱甘肽S-转移酶融合蛋白）与其RNA靶标Rev反应元件之间存在特异性交联。不可能在RNA噬菌体MS2外壳蛋白与其结合的复制酶基因的起始茎环之间产生交联。这些结果与两个系统现有的结构数据一致。
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Preparation of oligoribonucleotides containing 4-thiouridine using Fpmp chemistry. Photo-crosslinking to RNA binding proteins using 350 nm irradiation.使用Fpmp化学方法制备含4-硫尿苷的寡核糖核苷酸。利用350纳米辐射对RNA结合蛋白进行光交联。
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