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High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme

Published online by Cambridge University Press:  01 September 1999


THOMAS P. SHIELDS
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA Present address: Department of Chemistry, Petty Science Building, University of North Carolina at Greensboro, Greensboro, North Carolina 27403, USA.
EMILIA MOLLOVA
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA
LINDA STE. MARIE
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA
MARK R. HANSEN
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA
ARTHUR PARDI
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA

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Abstract

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.


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Copyright
© 1999 RNA Society

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