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

1999; Cold Spring Harbor Laboratory Press; Volume: 5; Issue: 9 Linguagem: Inglês

10.1017/s1355838299990945

1469-9001

Thomas P. Shields, Emilia T. Mollova, L. Marié, Mark R. Hansen, Arthur Pardi,

Advanced biosensing and bioanalysis techniques

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.