Improved native affinity purification of RNA

被引:97
作者
Batey, Robert T.
Kieft, Jeffrey S.
机构
[1] Univ Colorado, Hlth Sci Ctr, Dept Biochem & Mol Genet, Aurora, CO 80045 USA
[2] Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA
关键词
RNA purification; affinity tag; activatable ribozyme; native purification;
D O I
10.1261/rna.528007
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable.
引用
收藏
页码:1384 / 1389
页数:6
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