Improved native affinity purification of RNA
Abstract
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.
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Footnotes
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Reprint requests to: Jeffrey S. Kieft, Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Mail Stop 8101, P.O. Box 6511, Aurora, CO 80045, USA; e-mail: Jeffrey.Kieft{at}uchsc.edu; fax: (303) 724-3257; Robert T. Batey, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 80309-0215, USA; e-mail: Robert.Batey{at}colorado.edu.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.528007.
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- Received February 28, 2007.
- Accepted April 16, 2007.
- Copyright © 2007 RNA Society
