RNA及核酶的合成、去保护、分析与纯化。
Synthesis, deprotection, analysis and purification of RNA and ribozymes.
作者信息
Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S, Usman N
机构信息
Department of Chemistry and Biochemistry, Ribozyme Pharmaceuticals Inc., Boulder, CO 80301, USA.
出版信息
Nucleic Acids Res. 1995 Jul 25;23(14):2677-84. doi: 10.1093/nar/23.14.2677.
Abstract
Improvements in the synthesis, deprotection and purification of oligoribonucleotides are described. These advances allow for reduced synthesis and deprotection times, while improving product yield. Coupling times are reduced by half using 5-ethylthio-1H-tetrazole (S-ethyltetrazole) as the activator. Base and 2'-O-t-butyldimethylsilyl deprotection with methylamine (MA) and anhydrous triethylamine/hydrogen fluoride in N-methylpyrrolidinone (TEA.HF/NMP), respectively, requires a fraction of the time necessitated by current standard methods. In addition, the ease of oligoribonucleotide purification and analysis have been significantly enhanced using anion exchange chromatography. These new methods improve the yield and quality of the oligoribonucleotides synthesized. Hammerhead ribozymes synthesized utilizing the described methods exhibited no diminution in catalytic activity.
摘要
本文描述了寡核糖核苷酸合成、脱保护及纯化方面的改进。这些进展使得合成和脱保护时间缩短，同时提高了产物收率。使用5-乙硫基-1H-四唑（S-乙基四唑）作为活化剂，偶联时间减少了一半。分别用甲胺（MA）和N-甲基吡咯烷酮中的无水三乙胺/氟化氢（TEA.HF/NMP）进行碱基和2'-O-叔丁基二甲基甲硅烷基脱保护，所需时间仅为当前标准方法的一小部分。此外，使用阴离子交换色谱法可显著提高寡核糖核苷酸纯化和分析的便利性。这些新方法提高了合成的寡核糖核苷酸的收率和质量。利用所述方法合成的锤头状核酶的催化活性未降低。
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