基于带电荷氨基酸α-NH2基团生物素化的氨酰-tRNA快速筛选。
Rapid selection of aminoacyl-tRNAs based on biotinylation of alpha-NH2 group of charged amino acids.
作者信息
Pütz J, Wientges J, Sissler M, Giegé R, Florentz C, Schwienhorst A
机构信息
Institüt für Molekulare Biotechnologie, Beutenbergstrasse 11, D-07745, Jena, Germany.
出版信息
Nucleic Acids Res. 1997 May 1;25(9):1862-3. doi: 10.1093/nar/25.9.1862.
Abstract
A rapid selection procedure to separate low amounts of aminoacylated tRNAs from large pools of inactive variants is described. The procedure involves a three-step protocol. After initial aminoacylation of a tRNA pool, N-hydroxysuccinimide ester chemistry is applied to biotinylate the alpha-NH2 group of the amino acid bound to the 3'-end of a tRNA. The biotin tag is used to capture the derivatized tRNAs on streptavidin-conjugated magnetic beads. Variants bound to the solid phase can be amplified by RT-PCR and transcription, providing tRNAs for subsequent selection rounds.
摘要
本文描述了一种从大量无活性变体中分离少量氨酰化tRNA的快速筛选方法。该方法包括一个三步方案。在对tRNA库进行初始氨酰化后，应用N-羟基琥珀酰亚胺酯化学方法对与tRNA 3'-末端结合的氨基酸的α-NH2基团进行生物素化。生物素标签用于在链霉亲和素偶联的磁珠上捕获衍生化的tRNA。与固相结合的变体可以通过RT-PCR和转录进行扩增，为后续的筛选轮次提供tRNA。
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本文引用的文献
1
2
Selection of aminoacylated tRNAs from RNA libraries having randomized acceptor stem sequences: using old dogs to perform new tricks.从具有随机化受体茎序列的RNA文库中选择氨酰化tRNA：让老狗学新把戏。
Methods Enzymol. 1996;267:384-410. doi: 10.1016/s0076-6879(96)67024-4.
3
Ribozyme-catalysed amino-acid transfer reactions.核酶催化的氨基酸转移反应。
Nature. 1996 May 30;381(6581):442-4. doi: 10.1038/381442a0.
4
Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP.通过固定化嗜热栖热菌EF-Tu.GTP亲和层析法纯化氨酰-tRNA
Anal Biochem. 1995 Jul 1;228(2):330-5. doi: 10.1006/abio.1995.1359.
5
Selection for active E. coli tRNA(Phe) variants from a randomized library using two proteins.使用两种蛋白质从随机文库中筛选活性大肠杆菌苯丙氨酸转运RNA（tRNA(Phe)）变体。
EMBO J. 1993 Jul;12(7):2959-67. doi: 10.1002/j.1460-2075.1993.tb05958.x.
6
Diversity of oligonucleotide functions.寡核苷酸功能的多样性。
Annu Rev Biochem. 1995;64:763-97. doi: 10.1146/annurev.bi.64.070195.003555.
7
Aminoacyl-RNA synthesis catalyzed by an RNA.由RNA催化的氨酰基-RNA合成。
Science. 1995 Feb 3;267(5198):643-7. doi: 10.1126/science.7530860.
8
Acylation of transfer ribonucleic acid with the N-hydroxysuccinimide ester of phenoxyacetic acid.用苯氧乙酸的N-羟基琥珀酰亚胺酯对转移核糖核酸进行酰化作用。
Biochemistry. 1972 Aug 29;11(18):3435-43. doi: 10.1021/bi00768a017.
9
tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.
Eur J Biochem. 1988 Mar 15;172(3):663-8. doi: 10.1111/j.1432-1033.1988.tb13940.x.
10
Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.指数富集的配体系统进化：噬菌体T4 DNA聚合酶的RNA配体
Science. 1990 Aug 3;249(4968):505-10. doi: 10.1126/science.2200121.
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