HPLC purification of RNA for crystallography and NMR

Abstract

Homogeneous preparations of milligram quantities of RNA are a prerequisite for their characterization by biophysical methods such as crystallography or NMR spectroscopy. Methods for obtaining milligram quantities of pure synthetic RNA are described in this paper. These methods employ anion exchange HPLC for purifying full-length sequence from failure sequences and incompletely deprotected material. RNA molecules with little or extensive amounts of secondary structure could be purified. In cases where the RNA molecule was tightly folded, the cation in the eluent buffer influenced both the distinction of the peaks during chromatography and the final folded conformation. Finally, two RNA sequences were chemically synthesized, deprotected, purified, and crystallized using this methodology.